CA1135183A - Dispersed metal particle labelled assay - Google Patents
Dispersed metal particle labelled assayInfo
- Publication number
- CA1135183A CA1135183A CA000331816A CA331816A CA1135183A CA 1135183 A CA1135183 A CA 1135183A CA 000331816 A CA000331816 A CA 000331816A CA 331816 A CA331816 A CA 331816A CA 1135183 A CA1135183 A CA 1135183A
- Authority
- CA
- Canada
- Prior art keywords
- component
- reagent
- reaction
- metal
- particles
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 239000002923 metal particle Substances 0.000 title claims 2
- 238000003556 assay Methods 0.000 title description 6
- 239000000306 component Substances 0.000 claims abstract description 329
- 239000002245 particle Substances 0.000 claims abstract description 215
- 229910052751 metal Inorganic materials 0.000 claims abstract description 142
- 239000002184 metal Substances 0.000 claims abstract description 142
- 238000000034 method Methods 0.000 claims abstract description 112
- 238000006243 chemical reaction Methods 0.000 claims abstract description 104
- 238000012360 testing method Methods 0.000 claims abstract description 77
- 239000006185 dispersion Substances 0.000 claims abstract description 60
- 239000000126 substance Substances 0.000 claims abstract description 39
- 150000002736 metal compounds Chemical class 0.000 claims abstract description 38
- 230000000875 corresponding effect Effects 0.000 claims abstract description 36
- 108091008324 binding proteins Proteins 0.000 claims abstract description 35
- 230000009870 specific binding Effects 0.000 claims abstract description 35
- 238000001514 detection method Methods 0.000 claims abstract description 26
- 229920000642 polymer Polymers 0.000 claims abstract description 24
- 230000008878 coupling Effects 0.000 claims abstract description 15
- 238000010168 coupling process Methods 0.000 claims abstract description 15
- 238000005859 coupling reaction Methods 0.000 claims abstract description 15
- 238000000926 separation method Methods 0.000 claims abstract description 10
- 230000000704 physical effect Effects 0.000 claims abstract description 9
- 230000035484 reaction time Effects 0.000 claims abstract description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 138
- 239000000243 solution Substances 0.000 claims description 112
- 230000000984 immunochemical effect Effects 0.000 claims description 103
- 108060003951 Immunoglobulin Proteins 0.000 claims description 102
- 102000018358 immunoglobulin Human genes 0.000 claims description 102
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 59
- 239000010931 gold Substances 0.000 claims description 54
- 229910052737 gold Inorganic materials 0.000 claims description 52
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 51
- 230000027455 binding Effects 0.000 claims description 47
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 43
- 102000004576 Placental Lactogen Human genes 0.000 claims description 42
- 108010003044 Placental Lactogen Proteins 0.000 claims description 42
- 239000000381 Placental Lactogen Substances 0.000 claims description 42
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 claims description 34
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 claims description 34
- 239000000427 antigen Substances 0.000 claims description 34
- 102000036639 antigens Human genes 0.000 claims description 34
- 108091007433 antigens Proteins 0.000 claims description 34
- 102000023732 binding proteins Human genes 0.000 claims description 34
- 239000007787 solid Substances 0.000 claims description 33
- 239000012736 aqueous medium Substances 0.000 claims description 30
- 230000008569 process Effects 0.000 claims description 29
- 239000011248 coating agent Substances 0.000 claims description 26
- 238000000576 coating method Methods 0.000 claims description 26
- 229960003604 testosterone Drugs 0.000 claims description 25
- 241001494479 Pecora Species 0.000 claims description 24
- 102000023108 LH Receptors Human genes 0.000 claims description 22
- 108010011942 LH Receptors Proteins 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 22
- 230000001900 immune effect Effects 0.000 claims description 21
- 229920002521 macromolecule Polymers 0.000 claims description 20
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 19
- 229940098773 bovine serum albumin Drugs 0.000 claims description 19
- 239000000872 buffer Substances 0.000 claims description 19
- 201000005404 rubella Diseases 0.000 claims description 19
- 239000004332 silver Substances 0.000 claims description 19
- 210000002966 serum Anatomy 0.000 claims description 17
- 229910052709 silver Inorganic materials 0.000 claims description 17
- 238000005406 washing Methods 0.000 claims description 13
- 150000002739 metals Chemical class 0.000 claims description 12
- 230000008859 change Effects 0.000 claims description 10
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 10
- 102000005962 receptors Human genes 0.000 claims description 9
- 108020003175 receptors Proteins 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 7
- 239000003999 initiator Substances 0.000 claims description 5
- 239000000178 monomer Substances 0.000 claims description 5
- 229910052697 platinum Inorganic materials 0.000 claims description 5
- 238000006116 polymerization reaction Methods 0.000 claims description 5
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 4
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims description 4
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 claims description 4
- 229910052802 copper Inorganic materials 0.000 claims description 4
- 239000010949 copper Substances 0.000 claims description 4
- -1 haptens Substances 0.000 claims description 4
- JKFYKCYQEWQPTM-UHFFFAOYSA-N 2-azaniumyl-2-(4-fluorophenyl)acetate Chemical compound OC(=O)C(N)C1=CC=C(F)C=C1 JKFYKCYQEWQPTM-UHFFFAOYSA-N 0.000 claims description 3
- 229910021612 Silver iodide Inorganic materials 0.000 claims description 3
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 claims description 3
- 239000012928 buffer substance Substances 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 208000006454 hepatitis Diseases 0.000 claims description 3
- 231100000283 hepatitis Toxicity 0.000 claims description 3
- 238000011065 in-situ storage Methods 0.000 claims description 3
- 239000011541 reaction mixture Substances 0.000 claims description 3
- 229940045105 silver iodide Drugs 0.000 claims description 3
- UKUVVAMSXXBMRX-UHFFFAOYSA-N 2,4,5-trithia-1,3-diarsabicyclo[1.1.1]pentane Chemical compound S1[As]2S[As]1S2 UKUVVAMSXXBMRX-UHFFFAOYSA-N 0.000 claims description 2
- XHCLAFWTIXFWPH-UHFFFAOYSA-N [O-2].[O-2].[O-2].[O-2].[O-2].[V+5].[V+5] Chemical compound [O-2].[O-2].[O-2].[O-2].[O-2].[V+5].[V+5] XHCLAFWTIXFWPH-UHFFFAOYSA-N 0.000 claims description 2
- 229910052782 aluminium Inorganic materials 0.000 claims description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 2
- 235000014413 iron hydroxide Nutrition 0.000 claims description 2
- NCNCGGDMXMBVIA-UHFFFAOYSA-L iron(ii) hydroxide Chemical compound [OH-].[OH-].[Fe+2] NCNCGGDMXMBVIA-UHFFFAOYSA-L 0.000 claims description 2
- XCAUINMIESBTBL-UHFFFAOYSA-N lead(ii) sulfide Chemical compound [Pb]=S XCAUINMIESBTBL-UHFFFAOYSA-N 0.000 claims description 2
- IPJKJLXEVHOKSE-UHFFFAOYSA-L manganese dihydroxide Chemical compound [OH-].[OH-].[Mn+2] IPJKJLXEVHOKSE-UHFFFAOYSA-L 0.000 claims description 2
- ADZWSOLPGZMUMY-UHFFFAOYSA-M silver bromide Chemical compound [Ag]Br ADZWSOLPGZMUMY-UHFFFAOYSA-M 0.000 claims description 2
- QXKXDIKCIPXUPL-UHFFFAOYSA-N sulfanylidenemercury Chemical compound [Hg]=S QXKXDIKCIPXUPL-UHFFFAOYSA-N 0.000 claims description 2
- 239000004408 titanium dioxide Substances 0.000 claims description 2
- 229910001935 vanadium oxide Inorganic materials 0.000 claims description 2
- 239000000523 sample Substances 0.000 claims 50
- 239000007790 solid phase Substances 0.000 claims 42
- 239000012488 sample solution Substances 0.000 claims 35
- 239000007791 liquid phase Substances 0.000 claims 33
- 230000008105 immune reaction Effects 0.000 claims 24
- 239000003446 ligand Substances 0.000 claims 14
- 229920001480 hydrophilic copolymer Polymers 0.000 claims 9
- 229920001477 hydrophilic polymer Polymers 0.000 claims 9
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims 8
- 229910052742 iron Inorganic materials 0.000 claims 4
- 239000007795 chemical reaction product Substances 0.000 claims 3
- 239000005749 Copper compound Substances 0.000 claims 2
- 150000001880 copper compounds Chemical class 0.000 claims 2
- 229910001922 gold oxide Inorganic materials 0.000 claims 2
- 239000000376 reactant Substances 0.000 claims 2
- 229910001923 silver oxide Inorganic materials 0.000 claims 2
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 claims 1
- 101000874347 Streptococcus agalactiae IgA FC receptor Proteins 0.000 claims 1
- 229910052804 chromium Inorganic materials 0.000 claims 1
- 239000011651 chromium Substances 0.000 claims 1
- VQWFNAGFNGABOH-UHFFFAOYSA-K chromium(iii) hydroxide Chemical compound [OH-].[OH-].[OH-].[Cr+3] VQWFNAGFNGABOH-UHFFFAOYSA-K 0.000 claims 1
- 102000014914 Carrier Proteins Human genes 0.000 abstract 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 55
- 239000008279 sol Substances 0.000 description 49
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 36
- 239000012153 distilled water Substances 0.000 description 35
- 239000011780 sodium chloride Substances 0.000 description 28
- 239000000562 conjugate Substances 0.000 description 27
- 238000002360 preparation method Methods 0.000 description 25
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 13
- 239000007983 Tris buffer Substances 0.000 description 11
- 230000031700 light absorption Effects 0.000 description 11
- 239000008363 phosphate buffer Substances 0.000 description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 10
- 239000007924 injection Substances 0.000 description 8
- 229940090044 injection Drugs 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000002953 phosphate buffered saline Substances 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 229920001223 polyethylene glycol Polymers 0.000 description 6
- 229910000027 potassium carbonate Inorganic materials 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 5
- 229920001213 Polysorbate 20 Polymers 0.000 description 5
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 238000005497 microtitration Methods 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 5
- 239000001509 sodium citrate Substances 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 238000009835 boiling Methods 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 231100000673 dose–response relationship Toxicity 0.000 description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 4
- 230000001681 protective effect Effects 0.000 description 4
- 229910052938 sodium sulfate Inorganic materials 0.000 description 4
- 235000011152 sodium sulphate Nutrition 0.000 description 4
- 229960000281 trometamol Drugs 0.000 description 4
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 3
- 239000007832 Na2SO4 Substances 0.000 description 3
- 239000004793 Polystyrene Substances 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 230000004520 agglutination Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000000951 immunodiffusion Effects 0.000 description 3
- 238000007918 intramuscular administration Methods 0.000 description 3
- 229920002223 polystyrene Polymers 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 3
- 229910000162 sodium phosphate Inorganic materials 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 3
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 3
- 235000019263 trisodium citrate Nutrition 0.000 description 3
- 229940038773 trisodium citrate Drugs 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- 241000710799 Rubella virus Species 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000001493 electron microscopy Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 229910021505 gold(III) hydroxide Inorganic materials 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 235000019799 monosodium phosphate Nutrition 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- JRKICGRDRMAZLK-UHFFFAOYSA-L persulfate group Chemical group S(=O)(=O)([O-])OOS(=O)(=O)[O-] JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 description 2
- 235000015320 potassium carbonate Nutrition 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 229910001112 rose gold Inorganic materials 0.000 description 2
- 238000005185 salting out Methods 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 235000011083 sodium citrates Nutrition 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 240000003550 Eusideroxylon zwageri Species 0.000 description 1
- 229910015400 FeC13 Inorganic materials 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241000726306 Irus Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108091006629 SLC13A2 Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 229910021502 aluminium hydroxide Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 238000003321 atomic absorption spectrophotometry Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 208000037516 chromosome inversion disease Diseases 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 210000004246 corpus luteum Anatomy 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 239000000727 fraction Substances 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000005906 menstruation Effects 0.000 description 1
- 229910000000 metal hydroxide Inorganic materials 0.000 description 1
- 150000004692 metal hydroxides Chemical class 0.000 description 1
- 229910044991 metal oxide Inorganic materials 0.000 description 1
- 150000004706 metal oxides Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229960000380 propiolactone Drugs 0.000 description 1
- 229940070376 protein Drugs 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 239000012857 radioactive material Substances 0.000 description 1
- 230000036647 reaction Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 239000007785 strong electrolyte Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/808—Automated or kit
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/815—Test for named compound or class of compounds
- Y10S436/817—Steroids or hormones
- Y10S436/818—Human chorionic gonadotropin
Abstract
ABSTRACT OF THE DISCLOSURE
The present invention relates to a method for the detection and/or determination of one or more components of the reaction between a specific binding protein and the correspond-ing bindable substance, in which one or more labelled compo-nents are used, that are obtained by coupling particles of a dispersion of a metal, metal compound or polymer nuclei, coated with a metal or metal compound, having a particle size of at least 5 nm, directly or indirectly to the desired component of said reaction. During the reaction or after an adequate reaction time, the physical properties and/or the amount of the metal and/or the formed metal containing agglomerate, is/are deter-mined in the test sample, or optionally after a separation of the bound and free metal-labelled components in one of the derived fractions by methods known by themselves.
The present invention relates to a method for the detection and/or determination of one or more components of the reaction between a specific binding protein and the correspond-ing bindable substance, in which one or more labelled compo-nents are used, that are obtained by coupling particles of a dispersion of a metal, metal compound or polymer nuclei, coated with a metal or metal compound, having a particle size of at least 5 nm, directly or indirectly to the desired component of said reaction. During the reaction or after an adequate reaction time, the physical properties and/or the amount of the metal and/or the formed metal containing agglomerate, is/are deter-mined in the test sample, or optionally after a separation of the bound and free metal-labelled components in one of the derived fractions by methods known by themselves.
Description
~13S~83 Ihe invention relates to a method for the detection and/or determination of one or more components of the reaction between a specific binding protein and the corresponding bind-able substance in an aqueous test sample, whilst applying the known binding affinity of such components for one another. Such components can be receptor proteins or immunochemical components, such as haptens, antigens or antibodies.
There are a large number of immunochemical methods known in which the presence of a certain immunological component is determined qualitatively and/or quantitatively by utilizing the mutual reactions between such components, such as the reac-tion between antigen and the antibody to that antigen.
In the study of such reactions for the demonstration and/or determination of the desired component, use may be made of aids, for example physical aids such as an electron micro-scope, or use may be made of a reagent which is provided with a marker or label which can be determined or demonstrated res-pectively in a lower concentration than the immunocomplex formed itself.
As examples of the category of qualitative immuno-chemical techniques there may be named the irnmunodiffusion method developed in 1948 by Ouchterlony and the variant thereof, immuno-electrophoresis, developed in 1953 by Grabar. The radial immunodiffusion method developed by Mancini in 1965 can be used quantitatively.
The immunodiffusion technique can be characterized as the placing of a thin layer of agar on a glass plate, after which two holes are made in the gel, a little distance apart.
The test liquid with antigen is introduced into one of the holes, and an antiserum into the other. As a result of the diffus;on of the two substances in the gel, these meet each other and c 1~35~33 form a visible precipitation line. Though this method is rela-tively simple, it does possess a nurnber of disadvantages, in particular the fact that the diffusion lasts for some time and that the results usually only provide a qualitative indication.
~he other immunochemical techniques also have their drawbacks, which apart from specific disadvantages generally consist of too long a duration for the test, a low sensitivity and/or the provision of only qualitative indications.
In addition to these non-labelled immunochemical techniques, a number of labelled techniques have been developed with the passage of the years, amongst which there may be named the haemagglutination test in which one of the components is bound to the surface of erythrocytes, the immunofluorescence technique, in which one of the components is labelled with a fluorescent compound (fluorophore), the radioimmunological de-termination developed by Yalow and Berson about 1959, in which, instead of a fluorophore, a radio-active atom or radio-active group is used as marker, and the most recent technique of enzyme-immunological determination, about which the first publications appeared in 1971 from two groups working independently, namely the Swedish investigators Engvall and Perlmann and the Dutch Schuurs and van Weemen. This last-named determination is in principle analogous to the known radio-immunological determina-tions, but with the difference that an enzyme is used as label instead of radio-active labelling, The much-used radio-irnmunological determination has indisputably great merit, but there are a number of substantial drawbacks associated with this method, such as the risk factor related to working with radio-active material, the high cost of reagents and apparatus, the poor stability of radio-active labelled reagents and the requirement that only qualified
There are a large number of immunochemical methods known in which the presence of a certain immunological component is determined qualitatively and/or quantitatively by utilizing the mutual reactions between such components, such as the reac-tion between antigen and the antibody to that antigen.
In the study of such reactions for the demonstration and/or determination of the desired component, use may be made of aids, for example physical aids such as an electron micro-scope, or use may be made of a reagent which is provided with a marker or label which can be determined or demonstrated res-pectively in a lower concentration than the immunocomplex formed itself.
As examples of the category of qualitative immuno-chemical techniques there may be named the irnmunodiffusion method developed in 1948 by Ouchterlony and the variant thereof, immuno-electrophoresis, developed in 1953 by Grabar. The radial immunodiffusion method developed by Mancini in 1965 can be used quantitatively.
The immunodiffusion technique can be characterized as the placing of a thin layer of agar on a glass plate, after which two holes are made in the gel, a little distance apart.
The test liquid with antigen is introduced into one of the holes, and an antiserum into the other. As a result of the diffus;on of the two substances in the gel, these meet each other and c 1~35~33 form a visible precipitation line. Though this method is rela-tively simple, it does possess a nurnber of disadvantages, in particular the fact that the diffusion lasts for some time and that the results usually only provide a qualitative indication.
~he other immunochemical techniques also have their drawbacks, which apart from specific disadvantages generally consist of too long a duration for the test, a low sensitivity and/or the provision of only qualitative indications.
In addition to these non-labelled immunochemical techniques, a number of labelled techniques have been developed with the passage of the years, amongst which there may be named the haemagglutination test in which one of the components is bound to the surface of erythrocytes, the immunofluorescence technique, in which one of the components is labelled with a fluorescent compound (fluorophore), the radioimmunological de-termination developed by Yalow and Berson about 1959, in which, instead of a fluorophore, a radio-active atom or radio-active group is used as marker, and the most recent technique of enzyme-immunological determination, about which the first publications appeared in 1971 from two groups working independently, namely the Swedish investigators Engvall and Perlmann and the Dutch Schuurs and van Weemen. This last-named determination is in principle analogous to the known radio-immunological determina-tions, but with the difference that an enzyme is used as label instead of radio-active labelling, The much-used radio-irnmunological determination has indisputably great merit, but there are a number of substantial drawbacks associated with this method, such as the risk factor related to working with radio-active material, the high cost of reagents and apparatus, the poor stability of radio-active labelled reagents and the requirement that only qualified
-2-113~i83 personnel be allowed to perform these determinations.
The enzyme-immunological determination method does not possess these disadvantages, but it is nevertheless desir-able that new estimation techniques be developed which are even more sensitive, may be performed more rapidly, can be more rea-dily automated and/or make it possible to estimate more than one immunocomponent at the same time.
Furthermore, there is a strong need for a reliable and practical assay for receptor proteins.
me present invention relates to a process for the detection and/or determination of one or more components of the reaction between a specific binding protein and the correspond-ing bindable substance in an aqueous test sample, whilst apply-ing the known binding affinity of such components for one another, which is characterized by the fact that one or more labelled components, obtained by coupling directly or indirectly the desired component of said reaction to particles of an aqueous dispersion of a metal, metal compound or polymer nuclei coated with a metal or metal compound, having a particle size of at least 5 nm, w~.ereby during the reaction or after an adequate reaction time, optionally after separation of the bound and free labelled components, the physical properties and/or the amount of the metal and/or the formed metal containing agglo-merate is/are determined in the test sample or one of the derived fractions, by methods known by themselves, which deter-mination provides a qualitative and/or quantitative indication of the component or components to be detected and/or determined.
Ihe process according to the invention is especially suited for the estimation of immunochemical components, such as haptens, antigen~ and antibodies.
The particles of the aqueous dispersion of a metal,
The enzyme-immunological determination method does not possess these disadvantages, but it is nevertheless desir-able that new estimation techniques be developed which are even more sensitive, may be performed more rapidly, can be more rea-dily automated and/or make it possible to estimate more than one immunocomponent at the same time.
Furthermore, there is a strong need for a reliable and practical assay for receptor proteins.
me present invention relates to a process for the detection and/or determination of one or more components of the reaction between a specific binding protein and the correspond-ing bindable substance in an aqueous test sample, whilst apply-ing the known binding affinity of such components for one another, which is characterized by the fact that one or more labelled components, obtained by coupling directly or indirectly the desired component of said reaction to particles of an aqueous dispersion of a metal, metal compound or polymer nuclei coated with a metal or metal compound, having a particle size of at least 5 nm, w~.ereby during the reaction or after an adequate reaction time, optionally after separation of the bound and free labelled components, the physical properties and/or the amount of the metal and/or the formed metal containing agglo-merate is/are determined in the test sample or one of the derived fractions, by methods known by themselves, which deter-mination provides a qualitative and/or quantitative indication of the component or components to be detected and/or determined.
Ihe process according to the invention is especially suited for the estimation of immunochemical components, such as haptens, antigen~ and antibodies.
The particles of the aqueous dispersion of a metal,
-3-, : :
~1351~3 .
metal compound or polymer nuclei, coated with a metal or metal compound have a particle size of at least 5 nm and preferably 10 to 100 nm. Dispersions with a particle size of 10 to 100 nm of the dispersed phase are usually sols, but other types of disper-sions are not excluded.
me component to be labelled is coupled directly or indirectly to the dispersed particles. Under coupling is under-stood any chemical or physical binding, such as binding via co-valent bonds, via hydrogen bridges, polar attraction and ad-10 sorption.
Whenever the term "metal sol particles" is used inthe text which follows, this is understood to signify particles of a sol, consisting of a metal, a metal compound or polymer nuclei coated with a metal or metal compound.
The use of metal sol particles, in particular those of gold sols, which are covered on the surface with antibodies, for the demonstration of the distribution of an antigen over a cell surface by means of scanning electron microscopy, in which the metal sol particles are used as contrast enhancing label, has already been described some years ago (see article by M.
Horisberger in Experientia, pages 1147-1149, 15th October 1975), but the application of dispersed particles, preferably metal sol particles as a label for an immunological component for an in vitro qualitative and quantitative determination of immunological components, such as haptens, antigens and antibodies, in an aqueous test medium has not previously been reported and has surprisingly proved to be possible.
The metal sol particle immunochemical technique ac-cording to the invention which has been developed can be not only more sensitive than the known radio- and enzyme-immuno techniques, but renders it furthermore possible to demonstrate . , :
~3S~3 and to determine more than one immunological component in the same test medium simultaneously by utilizing sol particles of different chemical composition as labels.
The metal sols may be of metals or metal compounds, such as metal oxides, metal hydroxides and metal salts or of polymer nuclei coated with metals or metal compounds. As ex-amples, there may be named the metals platinum, gold, silver and copper, and the metal compounds, silver iodide, silver bro-mide, copper hydrous oxide, iron oxide, iron hydroxide or hy-drous oxide, aluminium hydroxide or hydrous oxide, chromiumhydroxide or hydrous oxide, vanadium oxide, arsenic sulphide, manganese hydroxide, lead sulphide, mercury sulphide, barium sulphate and titanium dioxide. In general all metals or metal compounds, which may be readily demonstrated by means of known techniques.
It is sometimes advantageous to use sols with dis-persed particles consisting of polymer nuclei, coated with the above mentioned metals or metal compounds. These particles have similar properties as the dispersed phase of pure metals or metal compound~, but size, density and metal content can be optimally combined.
Use is preferably made of metals or metal compounds which are not or in very small quantities present in the test medium, in particular of those metals or metal compounds which have a low detection limit in suitable analysis techniques.
The metal sol particles to be used as label may be prepared in a large number of ways which are in themselves known. For example, for the preparation of a gold sol reference is made to an article by G. Frens in Nature Physical Science 30 241, 20 (1973).
The metal ~ol particles carry a charge which confers a stabilizing effect by mutual repulsion. By addition of chief-ly strong electrolytes, the charge pattern is changed, as a result of which aggregation and flocculation occurs. This may be prevented by coating the particles with macromolecules possessing polar groups, such as proteins, polyethylene glycols, polymeric carbohydrates, polyvinyl alcohols and similar.
As protective proteins, it is possible to use anti-gens, antibodies and anti-antibodies, or immunochemically active frag~ents thereof, or haptens that have been coupled to immuno-chemically inert, protective macromolecules, which results di-rectly in immuno-components labelled with metal sol particles.
It is not necessary that immuno components are used exclusively for the coating of the metal sol particles, since though this does provide a stabilizing effect, the immunoche-mical reactivity may be less than expected, probably due to steric hindrance. It has therefore also proved advantageous to coat only partially with an immunocomponent, and to complete the coating with another protective, but immunochemically inert, material, such as inert proteins, for example albumin, a poly-ethylene glycol, or another polar macromolecule. As coating ma-terial, use may also be made of protein A or related proteins, which possess reactivity with respect to the Fc part of anti-bodies. After the coating of the metal sol particles with pro-tein A, a further coating may be brought about with a selected antibody.
Another possibility consists of first coating the metal sol particles with an inert hydrophilic macromolecule, e.g. a polymer or co-polymer, after which the immunological com-ponent is coupled to the coating material by adsorption or by covalent binding.
The globules obtained after coating may contain a ~135~?3 single metal sol particle, but it is also possible that the polymer encloses more than one metal sol particle.
The coating of the metal sol particles by the inert polymer may take place in two ways, either by bringing the metal sol into contact with the polymer or by introducing the metal sol into an environment containing a monomer, or various mono-mers, and causing these to polymerize or co-polymerize respect-ively in situ. The polymerization can be initiated under the influence of radiation, or by the addition of initiators, such as a persulphate.
- The coating of a metal sol particle by polymerization of the monomeric medium in which the particle is located, under the influence of an inorganic initiator such as a persulfate, meets with practical difficulties, since the sol flocculates on addition of such an initiator. It has now been found that such a coating is however possible by first protecting the metal sol particles, then introducing the protected particles into the monomeric medium, and after that finally initiating the polymer-ization. me compounds mentioned above merit consideration as protective material.
The components labelled with metal sol particles are used as a reagent, generally in combination with other reagents for the demonstration and quantification of receptor proteins and haptens, antigens and antibodies in an aqueous test medium, for which all sorts of immunochemical techniques, in use in radio-immuno and enzyme-immuno tests, receive consideration.
me invention therefore also relates to test-kits for use in such immunochemical techniques, which contain as the most important component a metal-labelled immunocomponent, consisting of a metal sol, the particles of which have either been directly coated by the desired immunocomponent, or have 1135~3 been coated by an inert macromolecule, onto which the immuno-component has be-en coupled or adsorbed.
One of the usual immunochemical techniques is the competitive immunotest, which can be used for the demonstration and estimation of every immunocomponent. For the demonstration, for example, of a certain antigen this method consists of bring-ing a test sample, containing an unknown quantity of antigen, into contact with either a certain amount of the metal-labelled antigen concerned and an insolubilized antibody ayainst this antigen, or a certain amount of insolubilized antigen and a metal-labelled antibody against this antigen.
After an adequate reaction time, the nature and/or the amount of the metal is determined in the bound or free frac-tion, which gives a qualitative or quantitative indication res-pectively of the antigen to be determined. With due changes in the details, an analogous method applies to the determination of other immunocomponents.
The so-called Sandwich techniques are also much used.
These are also particularly suitable for the use of a metal-labelled component according to the invention. According tothese techniques, an immunological component, for example an antibody if an antigen has to be determined, is rendered insolu-ble by coupling to a solid carrier. This solid carrier is for example the interior surace of the reaction vessel in which the immunochernical reaction is performed. After an initial incuba-tion, optionally followed by a washing step, a second incuba-tion with a metal-labelled antibody takes place, after which the metal is determined in the bound or the free phase. It is ad-vantageous to determine the metal in the bound phase. In this 3~ case after the separation of phases, the bound label is prefer-ably disengage~ so that the metal can be determined in the ob-,, --~3--,~
S~
tained liquid.
Furthermore, a homogeneous agglutination can be used.If the test sample contains the immunochemical component to be determined, agglutination occurs with the labelled immunochemi-cal component, resulting in a change of optical properties. A
qualitative or semi-quantitative result can be obtained by ob-servation of the colour change. It is also possible to observe the coarse of the reaction with a spectrophotometer.
In addition to the above-noted techniques, there are numerous other immunochemical techniques in which the metal-labelled immunocomponents can be used as reagent. 'rhe present invention also makes it possible to demonstrate different hap-tens, antigens, antibodies or combinations thereaf at the same time in one test sample, by using as reagent for each of the com-ponents to be demonstrated an immunocomponent which has been labelled with a different metal sol particle.
me measurement of the physical properties and/or the concentration of the metal and/or the formed metal containing agglomerate in a certain phase of the reaction mixture may take place u~ing numerous techniques, which are in themselves known.
As examples of these techniques there may be cited the colorime-tric determination, in which use is made of the intense colour of some dispersions which furthermore change colour with physi-cochemical changes, the visual method, which is often already applicable to qualitative determinations in view of the above-noted fact that metal sols are coloured, the use of flame emission spectrophotometry or another plasma-emission spectro-photometric method which renders simultaneous determination possible, and the highly sensitive method of flame-less atomic absorption spectrophotometr~.
'rhe invention is also related to test ki-ts to be 11351~3 used for the determination of one or more components of the re-action between a specific binding protein and the corresponding bindable substance in an aqueous medium, containing:
a) a metal labelled component which has been obtained by coupling a component of said reaction to particles of an aqueous dis-persion of a metal, metal compound or polymer nuclei coated with a metal or metal compound, which particles have a size of at least 5 nm;
b) other reagents.
Preferably the test kit contains a metal labelled com-ponent, consisting of a sol, having particles with a size varying from 10-100 nm, which are either directly coated by the compo-nent of said reaction or by an inert polymer, to which the de-sired component has been coupled.
The metal-labelled component of the test kit accord-ing to the invention can be a metal-labelled immunocomponent.
me metal-labelled component can be present as a dispersion, but it appeared to be surprisingly possible to obtain a stable freeze-dried product, that can be redispersed before use. ~he freeze-dried metal-labelled component can also contain the buffer substance, that is required for carrying out the reaction between the specific binding protein and the corresponding bind-able substance.
The invention is further illustrated by means of the following examples.
Example I - Colorimetric determination of human placental lacto-gen (HPL) with the aid of an antibody, labelled with gold par-ticles, from a rabbit anti-HPL serum.
1.1. Preparation of the qold sol 500 ml of a 0.10 g/l chloro-auric acid (HAUC14) so-lution in distilled water is heated to boiling point in a beaker ~13S~83 of 800 ml capacity. 3.5 ml of a 10 g/l solution of tri-sodium citrate in distilled water is introduced into the boiling solu-tion, after which the gold sol, which has become dark red after an initial blue colour, is boiled for a further 15 minutes. Af-ter cooling to room temperature, the red gold sol thus obtained is made up to a volume of 500 ml with distilled water in a volu-metric flask. The gold sol obtained in this way consists of gold particles with diameters between 45 and 70 nm, as verified by electron microscopy. A gold sol prepared in this way has a pH of 3.45 - 0.07 and a light-absorption maximum at 536 nm, While 536 nm 1.2. Preparation of rabbit anti-HPL sera Anti-HPL sera were prepared by injecting an HPL solu-tion into rabbits according to the following scheme:
Day Method of ~ g HPL Dissolved in with CFA
iniection NaCl 9 a/l 1intramuscular 100 0.5 ml 0.5 ml " 200 0.5 ml 0.5 ml 29 " 400 0.5 ml 0.5 ml 20 43intravenous 200 1.0 ml blood sample for determination of the titre of the anti-~erum in an EIA test.
me rabbits were bled out as soon as the titre of the antiserum in an EIA HPL test was greater than 1:5000. If this was not the case, the rabbits received an additional injec-tion of 200 ~g HPL in 1.0 ml 9 g/l NaCl (intravenous) and the titre of the antiserum was determined again 7 days after this injection. The antisera were stored at -25C or freeze-dried and then stored as a dry powder at -25C.
1.3. Preparation of the anti-HPL immune qlobulin solution from the rabbit anti-HPL serum.
.
~135~
For this purpose, 600 mg freeæe-dried rabbit anti-HPL serum is dissolved in 7.5 ml of an 9 g/l NaCl solution. 10 ml 180 g/l ~a2SO4 solution in distilled water is then added, followed by 1.35 g solid Na2S04. The turbid liquid obtained is allowed to stand in a centrifuge tube for 1 hour, after which the liquid is centrifuged at 25000 N/kg for 10 minutes. The supernatant is sucked of~ and the residue is redispersed in 20 ml of an 180 g/l Na2S04 solution in distilled water. This pro-cedure is repeated twice.
After the last centrifugation and removal of the supernatant by suction, the residue is dissolved in 20 ml of a 9 g/l solution of NaCl in distilled water. Thi5 solution is dia-lysed against 6 litres dialysis liquid consisting of a 0.3 g/l NaCl solution in distilled water, which has been adjusted to a pH of 7.0 with a solution of 0.2 mol K2CO3 in 1 litre distilled water. Dialysis is continued for 16 hours at 4C, after which the dialysed liquid is centrifuged at 160000 N/kg for 20 minutes.
The immunoglobulin solution is stored in 1 ml vials at a tempe-rature of -20C.
The immunoglobulin content is determined by measuring A1260 nm and A180 nm of a tenfold dilution of the immunoglobulin solution, using a spectrophotometer. Ihe immunoglobulin con-tent G in mg/ml can then be calculated using the formula G = 10 x [(1.45 x A280 nm)~(0-75 x A260 nm)]-1.4. Preparatlon of the qold particle - rabbit anti~HPL
_mrnunoqlobulin coniuqate.
500 ml of the gold sol prepared in the way described under 1.1. is adjusted to pH 7.0 by means of a solution of 0.2 mol K2C03 in 1 litre distilled water. 0.5 ml of the rabbit anti-HPL immunoglobulin solution, with a content of 125~ g immu-noylobulin pcr ml, is acldecl dropwise with vigorous stirring to 1~35183 25 ml of the neutralized gold sol. 0.5 ml of a 50 g/l solution of bovine serum albumin (BSA) in 5 mmol NaCl/litre is distilled water, which has been adjusted to a pH of 7.0 with a 0.2 mol K2CO3 solutlon in 1 litre water, is then added, also with stir-ring.
The ~old particle-rabbit anti-HPL immunoglobulin con-jugate obtained in this way is centrifuged at 25000 N/kg for 10 minutes, after which the supernatant liquid is sucked off. The red pellet, consisting of the conjugate, is taken up in such a volume (about 23 ml) of trometamol, 0.01 mol/litre adjusted to a pH of 7.4 with 0.01 mol/litre HCl in distilled water (0.01 mol/litre TRIS/HCl buffer, pH = 7.4), that the final A536Cmnm is 1.00.
1.5. Coatinq of Microelisa (R) plates with rabbit anti-HPL
immunoalobulins.
To this end a rabbit anti-HPL immunoglobulin solution is prepared with a content of 25~ g immunoglobulin per ml, in a solution of 0.04 mol Na2HPO4 per litre adjusted to a pH of 7.4 with a solution of 0.04 mol NaH2PO4 per litre, both in distilled water, to which 0.1 g/l Merthiolate has been added.
0.1 ml of the above-described immunoglobulin solution iR placed in each well of the Microelisa(R) plate, after which the plate is incubated for 16 hours at 0 - 4C. 0.1 ml 200 g/l BSA Rolution in a 0.04 mol/l phosphate buffer, pH 7.4, to which 0.1 g/l Merthiolate has been added, is then pipetted into each well, and the whole is incubated for a further 30 minutes at room temperature. The wells of the Microelisa( ) plates are now sucked empty and washed 3 times with distilled water, after which khey are kept at -~.0C until use.
1.6. Test Protocol for HPL estimation with qold particle rabbit anti-HPL immunoqlobulin coniu~ate.
!: 13 il351~3 1.6.a. Determination of a standard curve for HPL
A standard curve was constructed for HPL according to the protocol below.
1. Pipette 0.1 ml of the standard HPL solution into a well of a Microelisa( ) plate coated with rabbit anti-HPL immunoglobulin and incubate for 2 hours at room temperature.
2. Suck the well empty and wash it with 0.1 ml 0.01 mol/l Tris/
- HCl buffer, pH 7.4.
3. Pipette 0.1 ml gold particle rabbit anti-HPL immunoglobulin conjugate (A136Cm = 1.00) into the well and incubate overnight at room temperature.
~1351~3 .
metal compound or polymer nuclei, coated with a metal or metal compound have a particle size of at least 5 nm and preferably 10 to 100 nm. Dispersions with a particle size of 10 to 100 nm of the dispersed phase are usually sols, but other types of disper-sions are not excluded.
me component to be labelled is coupled directly or indirectly to the dispersed particles. Under coupling is under-stood any chemical or physical binding, such as binding via co-valent bonds, via hydrogen bridges, polar attraction and ad-10 sorption.
Whenever the term "metal sol particles" is used inthe text which follows, this is understood to signify particles of a sol, consisting of a metal, a metal compound or polymer nuclei coated with a metal or metal compound.
The use of metal sol particles, in particular those of gold sols, which are covered on the surface with antibodies, for the demonstration of the distribution of an antigen over a cell surface by means of scanning electron microscopy, in which the metal sol particles are used as contrast enhancing label, has already been described some years ago (see article by M.
Horisberger in Experientia, pages 1147-1149, 15th October 1975), but the application of dispersed particles, preferably metal sol particles as a label for an immunological component for an in vitro qualitative and quantitative determination of immunological components, such as haptens, antigens and antibodies, in an aqueous test medium has not previously been reported and has surprisingly proved to be possible.
The metal sol particle immunochemical technique ac-cording to the invention which has been developed can be not only more sensitive than the known radio- and enzyme-immuno techniques, but renders it furthermore possible to demonstrate . , :
~3S~3 and to determine more than one immunological component in the same test medium simultaneously by utilizing sol particles of different chemical composition as labels.
The metal sols may be of metals or metal compounds, such as metal oxides, metal hydroxides and metal salts or of polymer nuclei coated with metals or metal compounds. As ex-amples, there may be named the metals platinum, gold, silver and copper, and the metal compounds, silver iodide, silver bro-mide, copper hydrous oxide, iron oxide, iron hydroxide or hy-drous oxide, aluminium hydroxide or hydrous oxide, chromiumhydroxide or hydrous oxide, vanadium oxide, arsenic sulphide, manganese hydroxide, lead sulphide, mercury sulphide, barium sulphate and titanium dioxide. In general all metals or metal compounds, which may be readily demonstrated by means of known techniques.
It is sometimes advantageous to use sols with dis-persed particles consisting of polymer nuclei, coated with the above mentioned metals or metal compounds. These particles have similar properties as the dispersed phase of pure metals or metal compound~, but size, density and metal content can be optimally combined.
Use is preferably made of metals or metal compounds which are not or in very small quantities present in the test medium, in particular of those metals or metal compounds which have a low detection limit in suitable analysis techniques.
The metal sol particles to be used as label may be prepared in a large number of ways which are in themselves known. For example, for the preparation of a gold sol reference is made to an article by G. Frens in Nature Physical Science 30 241, 20 (1973).
The metal ~ol particles carry a charge which confers a stabilizing effect by mutual repulsion. By addition of chief-ly strong electrolytes, the charge pattern is changed, as a result of which aggregation and flocculation occurs. This may be prevented by coating the particles with macromolecules possessing polar groups, such as proteins, polyethylene glycols, polymeric carbohydrates, polyvinyl alcohols and similar.
As protective proteins, it is possible to use anti-gens, antibodies and anti-antibodies, or immunochemically active frag~ents thereof, or haptens that have been coupled to immuno-chemically inert, protective macromolecules, which results di-rectly in immuno-components labelled with metal sol particles.
It is not necessary that immuno components are used exclusively for the coating of the metal sol particles, since though this does provide a stabilizing effect, the immunoche-mical reactivity may be less than expected, probably due to steric hindrance. It has therefore also proved advantageous to coat only partially with an immunocomponent, and to complete the coating with another protective, but immunochemically inert, material, such as inert proteins, for example albumin, a poly-ethylene glycol, or another polar macromolecule. As coating ma-terial, use may also be made of protein A or related proteins, which possess reactivity with respect to the Fc part of anti-bodies. After the coating of the metal sol particles with pro-tein A, a further coating may be brought about with a selected antibody.
Another possibility consists of first coating the metal sol particles with an inert hydrophilic macromolecule, e.g. a polymer or co-polymer, after which the immunological com-ponent is coupled to the coating material by adsorption or by covalent binding.
The globules obtained after coating may contain a ~135~?3 single metal sol particle, but it is also possible that the polymer encloses more than one metal sol particle.
The coating of the metal sol particles by the inert polymer may take place in two ways, either by bringing the metal sol into contact with the polymer or by introducing the metal sol into an environment containing a monomer, or various mono-mers, and causing these to polymerize or co-polymerize respect-ively in situ. The polymerization can be initiated under the influence of radiation, or by the addition of initiators, such as a persulphate.
- The coating of a metal sol particle by polymerization of the monomeric medium in which the particle is located, under the influence of an inorganic initiator such as a persulfate, meets with practical difficulties, since the sol flocculates on addition of such an initiator. It has now been found that such a coating is however possible by first protecting the metal sol particles, then introducing the protected particles into the monomeric medium, and after that finally initiating the polymer-ization. me compounds mentioned above merit consideration as protective material.
The components labelled with metal sol particles are used as a reagent, generally in combination with other reagents for the demonstration and quantification of receptor proteins and haptens, antigens and antibodies in an aqueous test medium, for which all sorts of immunochemical techniques, in use in radio-immuno and enzyme-immuno tests, receive consideration.
me invention therefore also relates to test-kits for use in such immunochemical techniques, which contain as the most important component a metal-labelled immunocomponent, consisting of a metal sol, the particles of which have either been directly coated by the desired immunocomponent, or have 1135~3 been coated by an inert macromolecule, onto which the immuno-component has be-en coupled or adsorbed.
One of the usual immunochemical techniques is the competitive immunotest, which can be used for the demonstration and estimation of every immunocomponent. For the demonstration, for example, of a certain antigen this method consists of bring-ing a test sample, containing an unknown quantity of antigen, into contact with either a certain amount of the metal-labelled antigen concerned and an insolubilized antibody ayainst this antigen, or a certain amount of insolubilized antigen and a metal-labelled antibody against this antigen.
After an adequate reaction time, the nature and/or the amount of the metal is determined in the bound or free frac-tion, which gives a qualitative or quantitative indication res-pectively of the antigen to be determined. With due changes in the details, an analogous method applies to the determination of other immunocomponents.
The so-called Sandwich techniques are also much used.
These are also particularly suitable for the use of a metal-labelled component according to the invention. According tothese techniques, an immunological component, for example an antibody if an antigen has to be determined, is rendered insolu-ble by coupling to a solid carrier. This solid carrier is for example the interior surace of the reaction vessel in which the immunochernical reaction is performed. After an initial incuba-tion, optionally followed by a washing step, a second incuba-tion with a metal-labelled antibody takes place, after which the metal is determined in the bound or the free phase. It is ad-vantageous to determine the metal in the bound phase. In this 3~ case after the separation of phases, the bound label is prefer-ably disengage~ so that the metal can be determined in the ob-,, --~3--,~
S~
tained liquid.
Furthermore, a homogeneous agglutination can be used.If the test sample contains the immunochemical component to be determined, agglutination occurs with the labelled immunochemi-cal component, resulting in a change of optical properties. A
qualitative or semi-quantitative result can be obtained by ob-servation of the colour change. It is also possible to observe the coarse of the reaction with a spectrophotometer.
In addition to the above-noted techniques, there are numerous other immunochemical techniques in which the metal-labelled immunocomponents can be used as reagent. 'rhe present invention also makes it possible to demonstrate different hap-tens, antigens, antibodies or combinations thereaf at the same time in one test sample, by using as reagent for each of the com-ponents to be demonstrated an immunocomponent which has been labelled with a different metal sol particle.
me measurement of the physical properties and/or the concentration of the metal and/or the formed metal containing agglomerate in a certain phase of the reaction mixture may take place u~ing numerous techniques, which are in themselves known.
As examples of these techniques there may be cited the colorime-tric determination, in which use is made of the intense colour of some dispersions which furthermore change colour with physi-cochemical changes, the visual method, which is often already applicable to qualitative determinations in view of the above-noted fact that metal sols are coloured, the use of flame emission spectrophotometry or another plasma-emission spectro-photometric method which renders simultaneous determination possible, and the highly sensitive method of flame-less atomic absorption spectrophotometr~.
'rhe invention is also related to test ki-ts to be 11351~3 used for the determination of one or more components of the re-action between a specific binding protein and the corresponding bindable substance in an aqueous medium, containing:
a) a metal labelled component which has been obtained by coupling a component of said reaction to particles of an aqueous dis-persion of a metal, metal compound or polymer nuclei coated with a metal or metal compound, which particles have a size of at least 5 nm;
b) other reagents.
Preferably the test kit contains a metal labelled com-ponent, consisting of a sol, having particles with a size varying from 10-100 nm, which are either directly coated by the compo-nent of said reaction or by an inert polymer, to which the de-sired component has been coupled.
The metal-labelled component of the test kit accord-ing to the invention can be a metal-labelled immunocomponent.
me metal-labelled component can be present as a dispersion, but it appeared to be surprisingly possible to obtain a stable freeze-dried product, that can be redispersed before use. ~he freeze-dried metal-labelled component can also contain the buffer substance, that is required for carrying out the reaction between the specific binding protein and the corresponding bind-able substance.
The invention is further illustrated by means of the following examples.
Example I - Colorimetric determination of human placental lacto-gen (HPL) with the aid of an antibody, labelled with gold par-ticles, from a rabbit anti-HPL serum.
1.1. Preparation of the qold sol 500 ml of a 0.10 g/l chloro-auric acid (HAUC14) so-lution in distilled water is heated to boiling point in a beaker ~13S~83 of 800 ml capacity. 3.5 ml of a 10 g/l solution of tri-sodium citrate in distilled water is introduced into the boiling solu-tion, after which the gold sol, which has become dark red after an initial blue colour, is boiled for a further 15 minutes. Af-ter cooling to room temperature, the red gold sol thus obtained is made up to a volume of 500 ml with distilled water in a volu-metric flask. The gold sol obtained in this way consists of gold particles with diameters between 45 and 70 nm, as verified by electron microscopy. A gold sol prepared in this way has a pH of 3.45 - 0.07 and a light-absorption maximum at 536 nm, While 536 nm 1.2. Preparation of rabbit anti-HPL sera Anti-HPL sera were prepared by injecting an HPL solu-tion into rabbits according to the following scheme:
Day Method of ~ g HPL Dissolved in with CFA
iniection NaCl 9 a/l 1intramuscular 100 0.5 ml 0.5 ml " 200 0.5 ml 0.5 ml 29 " 400 0.5 ml 0.5 ml 20 43intravenous 200 1.0 ml blood sample for determination of the titre of the anti-~erum in an EIA test.
me rabbits were bled out as soon as the titre of the antiserum in an EIA HPL test was greater than 1:5000. If this was not the case, the rabbits received an additional injec-tion of 200 ~g HPL in 1.0 ml 9 g/l NaCl (intravenous) and the titre of the antiserum was determined again 7 days after this injection. The antisera were stored at -25C or freeze-dried and then stored as a dry powder at -25C.
1.3. Preparation of the anti-HPL immune qlobulin solution from the rabbit anti-HPL serum.
.
~135~
For this purpose, 600 mg freeæe-dried rabbit anti-HPL serum is dissolved in 7.5 ml of an 9 g/l NaCl solution. 10 ml 180 g/l ~a2SO4 solution in distilled water is then added, followed by 1.35 g solid Na2S04. The turbid liquid obtained is allowed to stand in a centrifuge tube for 1 hour, after which the liquid is centrifuged at 25000 N/kg for 10 minutes. The supernatant is sucked of~ and the residue is redispersed in 20 ml of an 180 g/l Na2S04 solution in distilled water. This pro-cedure is repeated twice.
After the last centrifugation and removal of the supernatant by suction, the residue is dissolved in 20 ml of a 9 g/l solution of NaCl in distilled water. Thi5 solution is dia-lysed against 6 litres dialysis liquid consisting of a 0.3 g/l NaCl solution in distilled water, which has been adjusted to a pH of 7.0 with a solution of 0.2 mol K2CO3 in 1 litre distilled water. Dialysis is continued for 16 hours at 4C, after which the dialysed liquid is centrifuged at 160000 N/kg for 20 minutes.
The immunoglobulin solution is stored in 1 ml vials at a tempe-rature of -20C.
The immunoglobulin content is determined by measuring A1260 nm and A180 nm of a tenfold dilution of the immunoglobulin solution, using a spectrophotometer. Ihe immunoglobulin con-tent G in mg/ml can then be calculated using the formula G = 10 x [(1.45 x A280 nm)~(0-75 x A260 nm)]-1.4. Preparatlon of the qold particle - rabbit anti~HPL
_mrnunoqlobulin coniuqate.
500 ml of the gold sol prepared in the way described under 1.1. is adjusted to pH 7.0 by means of a solution of 0.2 mol K2C03 in 1 litre distilled water. 0.5 ml of the rabbit anti-HPL immunoglobulin solution, with a content of 125~ g immu-noylobulin pcr ml, is acldecl dropwise with vigorous stirring to 1~35183 25 ml of the neutralized gold sol. 0.5 ml of a 50 g/l solution of bovine serum albumin (BSA) in 5 mmol NaCl/litre is distilled water, which has been adjusted to a pH of 7.0 with a 0.2 mol K2CO3 solutlon in 1 litre water, is then added, also with stir-ring.
The ~old particle-rabbit anti-HPL immunoglobulin con-jugate obtained in this way is centrifuged at 25000 N/kg for 10 minutes, after which the supernatant liquid is sucked off. The red pellet, consisting of the conjugate, is taken up in such a volume (about 23 ml) of trometamol, 0.01 mol/litre adjusted to a pH of 7.4 with 0.01 mol/litre HCl in distilled water (0.01 mol/litre TRIS/HCl buffer, pH = 7.4), that the final A536Cmnm is 1.00.
1.5. Coatinq of Microelisa (R) plates with rabbit anti-HPL
immunoalobulins.
To this end a rabbit anti-HPL immunoglobulin solution is prepared with a content of 25~ g immunoglobulin per ml, in a solution of 0.04 mol Na2HPO4 per litre adjusted to a pH of 7.4 with a solution of 0.04 mol NaH2PO4 per litre, both in distilled water, to which 0.1 g/l Merthiolate has been added.
0.1 ml of the above-described immunoglobulin solution iR placed in each well of the Microelisa(R) plate, after which the plate is incubated for 16 hours at 0 - 4C. 0.1 ml 200 g/l BSA Rolution in a 0.04 mol/l phosphate buffer, pH 7.4, to which 0.1 g/l Merthiolate has been added, is then pipetted into each well, and the whole is incubated for a further 30 minutes at room temperature. The wells of the Microelisa( ) plates are now sucked empty and washed 3 times with distilled water, after which khey are kept at -~.0C until use.
1.6. Test Protocol for HPL estimation with qold particle rabbit anti-HPL immunoqlobulin coniu~ate.
!: 13 il351~3 1.6.a. Determination of a standard curve for HPL
A standard curve was constructed for HPL according to the protocol below.
1. Pipette 0.1 ml of the standard HPL solution into a well of a Microelisa( ) plate coated with rabbit anti-HPL immunoglobulin and incubate for 2 hours at room temperature.
2. Suck the well empty and wash it with 0.1 ml 0.01 mol/l Tris/
- HCl buffer, pH 7.4.
3. Pipette 0.1 ml gold particle rabbit anti-HPL immunoglobulin conjugate (A136Cm = 1.00) into the well and incubate overnight at room temperature.
4. Empty the well by suction and wash it with 0.3 ml 0.01 mol/l Tris/HCl/~aCl buffer, pH 7.4.
5. Pipette 0.1 ml 0.1 mol/1 HCl solution in water into the well and allow this to act for 30 minutes in the shaker.
6. Measure the light absorption at 536 nm with a small-volume spectrophotometer.
The results of the determination of a standard curve are given in the table below.
20 HPL concentration in the standard _53~, ^t t~L~5G
olution, na/ml the test 0 0.030 0.2 0.030 0.8 0.060 3.2 0.106 12.5 0.136 0.188 200 0.220 The solvent for the HPL standard solution is 0.04 mol/l phos-phate buffer + 1 g/l BSA + 9 g/l NaCl, pH 7.4.
1.6.b. Determination of HPL in the serum of pre~nant women.
The sera of pregnant women were diluted such that the HPL level ranged from 5 to 80 ng/ml.
'me diluent for the sera was 0.04 mol/l phosphate buffer, pH 7.4 ~ 1 g/l BSA + 9 g/l NaCl.
With the aid of the test protocol described under 1.6.a., the HPL concentrations of the diluted sera were deter-mined using the standard curve constructed. After correction for the dilution factor, the following results were obtained.
Serum HPL content, EIA test HPL content, MIA
A 4.5 + 0.5 ~g~ ml 4 - 1 ~g~ ml B 3.2 - 0.4 ~g/mll 3.5 - 0.8 ~g/ml C 0.7 + 0.1 ~g/ml 1 + 0.3 ~g/ml EXAMPLE II - Determination of HPL with the aid of gold-particle-labelled antibodies and atomic absorption spectrophotometry.
2.1. Pre~aration of reaqents and coated Microelisa plates The method of preparation of the gold particle rabbit anti-HPL immunoglobulin conjugate and the Microelisa(R) plates coated with rabbit anti-HPL immunoglobulin was the same as des-cribed in Example I.
2.2. Test protocol 2.2.a Determination of standard curve for HPL
In order to construct a standard curve for HPL, ~erial dilutions of HPL were made of consecutively 1, 10, 100 and 1000 ng/ml and a blank. me solvent for HPL was 0.04 mol/l phosphate buffer, pH 7.4, to which 1 g/l BSA and 9 g/l NaCl had been added.
1. Pipette 0.1 ml standard HPL solution into the well of the coated Microelisa plate and incubate for 2 hours at room tem-perature.
2. Suck the well empty and wash with 0.1 ml 0.02 mol/l Tris/HCl buffer, pH 7,~, .~! -15-1~3S1~33 3. Pipette 0.1 ml of the bold particle anti-HPL antibody conju-gate (A136Cmnm = 1.00) into the well and incubate this overnight (about 16~hours~.
4. Suck the well empty and wash it with 0.02 mol/l Tris buffer, pH 7.4.
5. Pipette 0.1 ml HCl, 0.1 mol/l, into the well and allow this to act with shaking on a shaking machine for 30 minutes.
6. ~ow measure the peak value of the light absorption at 242.8 nm in a flame atomic absorption spectrophotometer fitted with a small-volume injector.
A typical example of a standard curve obtained in this way is shown in the table below.
HPL concentration in Peak value of light absorption standard solution at 242.8 nm after termination of the test 0 ng/ml 0.020 1 ng/ml 0.040 10 ng/ml 0.160 100 ng/ml 0.200 201000 ng/ml 0.220 2.2.b. Determination of HPL in the serum of preqnant women.
The sera of pregnant women were diluted such that the HPL level lay in the range 5 - 80 ng/ml. The diluen-t for the sera was 0.04 mol/l phosphate buffer (pH = 7.4) + 1 g/1 BSA
+ 9 g/l NaCl. With the aid of the test protocol described under 2.2.a., the HPL concentration~ of the diluted sera were deter-mined using the standard curve constructed.
EXAMPLE III - Visual detection of hepatitis Bs antigen (HBsAg) by means of gold particle sheep anti-HBs immunoglobulin conju-gate.
3.1. Preparation of the qold sol ' -16-~13~ 3 See example 1.1.
3.2. Preparation of sheep anti-HBs sera Sheep were in~ected with purified HBsAg solution.
3.3. Preparation of the sheep anti-H~s immunoqlobulin solution 1.4 g solid Na2SO4 is added to 10 ml sheep anti-HBsAg serum. After all the sodium sulphate has dissolved, the turbid liquid is allowed to stand for 1 hour at room tempera-ture. ~he liquid is then centrifuged at 25000 N/kg for 10 mi-nutes. me supernatant is removed by suction and the residue is redispersed in 10 ml 140 g/l Na2SO4 solution in distilled water. This procedure is repeated twice.
After centrifuging for the last time and removing the supernatant by suction, the pellet is dissolved in 10 ml of 9 g/1 ~aC1 solution in distilled water. mis solution is dialysed against 6 litres of a 0.3 g/l NaCl solution in dis-tilled water, adjusted to a pH of 7.0 with a solution of 0.2 mol/l K2C03. Dialysis takes place for 16 hours at 4C, after which the dialysed liquid is centrifuged for 20 minutes at 160000 N~kg.
The immunoglobulin solution is stored in portions of 1 ml in vials at -20C. The immunoglobulin content is deter-mined by the A16oCmnm, A18Comnm method already described.
3.4. Pre~aration of the qold-particle sheep anti-HBs immuno-alobulin con1~qate.
The preparation of the conjugate is identical with the method of preparation described in example I point 4, with the exception that a diluted solution (also 125 ng immunoglo-bulin per ml) of the sheep anti-HBs immunoglobulin solution was used instead of the rabbit anti-HPL immunoglobulin solution.
3.5. Coatina of Microelisa( ) plates with sheeP anti-HBs mmunoalobulins : .. - .....
~35~
The coating of Microelisa( ) plates with sheep anti-HBs immunoglobulins is performed in the way described in example I point 5, whereby the sheep anti-HBs immunoglobulin solution was used instead of a rabbit anti-HPL immunoglobulin solution. Ten control wells were included in each plate. These wells were coated with an immunoglobulin solution from human serum which was negative in the following tests:
- Hepanosticon and Hepanostika - Monosticon - Rheumanosticon - Immunodiffusion against normal sheep serum - EIA for anti-HBs.
3.6. Test protocol for a visually readable test.
1. Pipette 0.1 ml of the sample into a well of the coated Micro-elisa plate and incubate for 2 hours at 37C.
2. Wash each well 3x with 0.3 ml 0.02 mol/l Tris/HCl buffer, pH 7.4.
3. Pipette 0.1 ml gold-particle sheep-anti-HBs conjugate 1A136m = 1.00) into the well and incubate overnight at room temperature.
~. Empty the well by suction and wash it 3x with 0.3 ml 0.01 mol/l Tris/HCl/NaCl buffer, pH 7.4.
5. Pipette 0.1 ml 0.1 mol/l HCl solution in distilled water into the well and allow this to act for 30 minutes, on a shaker.
6. A3~ess visually the colour of the liquid in the well and compare it with the control wells present.
Sera which gave a strong positive reaction in the Hepanostika test could generally still be distinguished from the controls in 8-fold dilution.
EXAMPLE IV - Determination of testosterone with the aid of a silver particle testosterone~ -succinyl-bovine serum albumin ~L~351~3 conjugate.
4.1._Preparation of the silver sol ~ .5 ml of a 10 g/l AgNO3 solution in distilled water is diluted with 486~5 ml distilled water and 5.0 ml of a 10 g/l solution of trisodium citrate.2H20 in distilled water is then added. 40.0 ml of a 10 g/l solution of hydrazine in distilled water is added to this solution at room temperature with vigorous stirring using a magnetic stirrer. A grey-yellow-green silver sol with a strong Tyndall scattering is formed within 60 seconds.
After 10-fold dilution with distilled water, the pH was ~.85 and the sol had a light absorption of A1416Cmnm = 0.83.
20 ml of the undiluted sol was centrifuged for 10 minutes at 25000 ~/kg. 15 ml of the supernatant was removed by suction and replaced by 15 ml of a 0.1 g/l trisodium citrate.
2H20 solution in distilled water (pH 6.88).
The washing process was repeated twice. After the final washing, the volume was made up to 20 ml with the 0.1 g/l sodium citrate solution.
After 4-fold dilution with distilled water, the sol 0 had the following properties: `
max = 416 nm 416 nm 1.12 pH = 7.0 4.~. Preparation of testosterone~ -hemisuccinate-BSA
40 mg testosterone-ll ~-hemisuccinate is dissolved in 2 ml dimethyl-formamide and the solution is cooled to -15C.
140 mg bovine serum albumin is dissolved in 3 ml distilled water, after which 1 drop 4N NaOH and 2 ml DMF are added, and the whole is cooled to -15C.
12.5 ~1 N-methylmorpholine and 12.5~11 isobutyl chloroformate are now added to the steroid solution. After 3 1135~33 minutes, the solution is added to the BSA solution. After stir-ring,for 1 hour at -15C and 3 hours at 0C, the solution is transferred to a dialysis bag and dialysed overnight against running tap water. The dialysate is passed through a Sephadex G25TM column, optionally after centrifugation, and the protein fraction is collected and freeze-dried.
4.3. Preparation of antisera for testosterone Rabbits were immunized by injection of 1.25 mg tes-tosterone-ll ~-hemisuccinyl-BSA, dissolved in 2.5 ml physio-logical saline, and 2.5 ml complete Freunds adjuvant (1 ml i.m. 3x, interval 1 week). They were then given an i.v. in-jection of 1 ml of a solution of 1.25 mg testosterone-ll~ -hemi-~uccinyl-BSA in 5 ml physiological saline.
This injection scheme was repeated until the titre of the antiserum was sufficiently high. The rabbits were then bled out, and the sera were stored at -20C until use.
4.4. Preparation of polystyrene tubes coated with rabbit immuno-~lobulin aqainst testosterone A rabbit anti-testosterone immunoglobulin solution is made by salting out with 180 g/l Na2S04 as described in example I point 2.
The dialysed immunoglobulin solution is diluted (to a concentration of 1~ g immunoglobulin/ml) with a buffer con-sisting of 0.1 mol/litre NaH2P04.H20, 60 g/l sucrose, adjusted to pH 7.4 with 4 mol/l NaOH. 1 ml of the immunoglobulin solu-tion i9 pipetted into each 3 ml polystyrene tube, and incubated overnight at room temperature. The tubes are then emptied by suction and filled with 1 ml 10 g/l BSA solution in the above-noted buffer. After two hours, the tubes are emptied by suc-tion and washed 3x with 3 ml distilled water. The tubes are dried completely by suction and are further drled over silica ~L1351S~3 gel overnight. They are then packed in aluminum foil in sets of 25 with a bag of silica gel and stored at 4C.
4.5. Preparation of silver particle-testosterone-ll ~-hemi-succinyl-BSA conjuqate 20 ml of the silver sol, prepared as described in 4.1., was diluted with 80 ml 0.3 g/l NaCl and the pH was adjusted to 7.0 with 0.01 mol/litre NaOH solution in distilled water. A421C nm = 1.08.
3 ml of a solution of testosterone-ll ~-hemisuccinyl-10 BSA, 175 ~ g/ml in 0.3 g/l NaCl adjusted to pH 7.0 with 0.01 mol/l NaOH, is then added dropwise to 15 ml of this sol with vigorous stirring by a magnetic stirrer.
The whole is incubated for about 10 minutes, afterwhich 2 ml 10 g/l Carbowax 20M solution in 0.3 g/l NaCl, adjusted to pH 7.0 with 0.01 mol/l NaOH, is added. The crude conjugate is subsequently washed by centrifuging 10 ml at 25000 N/kg for 20 minutes. 9 ml supernatant is now removed by suction and re-placed by 9 ml washing liquid consisting of 1 g/l Carbowax 20M
solution in 0.3 g/l ~aCl, adjusted to pH 7.0 with 0.01 mol/litre NaOH dissolved in distilled water. After washing 3 times, the conjugate is again centrifuged after which the supernatant liquid is removed by suction. me residue is redispersed in 0.02 mol/litre phosphate buffer, pH 7.4. me conjugate solution now has a pH of 7.4 and A421C nm = 1.08. The washed conjugates are stored at 4C until use.
4.6. Test protocol for the determination of testosterone with the silver particle testosterone-ll ~-hemisuccinyl-BSA coniuqate 1. Pipette a 1 ml sample into a polystyrene tube coated with rabbit anti-testosterone immunoglobulin and incubate for 2 hours at room temperature.
2. Empty the tube by suction and wash 3x with 2 ml 0.02 * - Trademark ~ l 1135~83 mol/litre phosphate buffer, pH 7Ø
3. Pipette 1 ml of the silver particle testosterone~
hemisuccinyl-BSA conju~ate, A42Clmnm = 1.08, into the tube and incubate for 16 hours.
4. Now suck the tube empty and wash with 2 ml 0.02 mol/litre phosphate buffer, p~ 7Ø
5. Pipette 1 ml 0.1 mol/litre HCl, solution in distilled water, into the tubes and allow this to act for 30 minutes.
6. Measure the liquid in the tube A121Cm 4.7. Results Ihe test media were colourimetrically determined with the aid of a standard curve, whereby amounts of testosterone of the magnitude of 0.5 nc3/ml and greater could be determined.
EX~PLE V - Determination of the titre of human anti-Rubella sera 5.1. Preparation of the Rubella anti~en 200 ml of a host cell suspension (BHK 21C3 from the Rijks Instituut voor Volksgezondheid, RIV) in culture medium (concentration 10 live cells per ml) is introduced into a roller bottle (surface 490 CM ). The cell culture is lcept for 16 to 20 hours at 37C. The culture medium is tnen removed and the cell layer is washed twice with 20 ml phosphate-buffered saline solution (PBS). 10 ml ~irus suspension (virus type ~33 RI~) is th~n diluted 1:100 with PBS and introduced into the bottle, after which the whole i~ :incubated at 37C for about 2 1/2 hours.
100 ml maintenance medium is now added and the cul-ture is then kept at 37C. After a period of at least 64 hours, but not exceeding 136 hours, depending on the assessment of the cytopathic effecl, the incubatecl mono]ayer of infected cells is harbe.sted. The mairltenarlce mediurrl is remove(l, and the mono-, . ...
11351~33 layer of cells is washed once with 20 ml PBS. The cells are loosened from the wall by means of sterile glass beads, taken up in 90 ml PBS and frozen at -20C.
After thawing, 10 ml glycine solution (1 mol/l), the pH being adjusted to 9.0 with solid NaOH, is added to 90 ml cell suspension, and the whole is thoroughly mixed for 6 hours at 37C. The cell suspension is now ultrascnicated for 30 minutes, with cooling in ice, and subsequently centrifuged at 30000 ~kg for 30 minutes at 4C. The supernatant containing the Rubella virus is collected and inactivated by consecutive addition of:
1:100 (v/v)NaOH (1 mol/litre) solution in distilled water 1:100 (v/v) freshly prepared 10%~ -propiolactone solution in distilled water, The liquid is allowed to stand for 48 hours at 4C, after which it is centrifuged for 30 minutes at 30000 N~kg and 4C.
me supernatant is the liquid containing the Rubella virus, which can if necessary be stored at -20C until use.
5.2. Coatinq of Microelisa( ) plates with Rubella antiaens.
The Rubella antigen solution, prepared as described in 5.1., is diluted 1:100 with a 0.05 mol/litre carbonate buffer, pH 9.6. 100 ~1 of this liquid Rubella antigen solution is pipetted into each well of the Microelisa plate and the whole is subsequently incubated for 16 hours at 4C. The well of the Microelisa(R) plate are now emptied by suction and washed 3 times with 300 ~1 0.05 mol/l phosphate buffer, pH 7.4, to which 9 g/l NaC1 and 0.5 g/l Tween 20 have been added.
5.3. Preparation of sheet anti-human immuno~lobulin solution Normal human serum is salted out with 140 g/1 Na2SO4, which i5 added as a solid in small portions. After standing for 1 hour at room temperature, the turbid liquid is centri-1~351~3 fuged at 25000 N~kg for 10 minutes. The supernatant is removed by suction, after which the residue is washed with a 140 g/l Na2S04 solution in distilled water. After centrifuging and removal of the supernatant by suction, the residue is dissolved in 9 g/l ~aCl, after which the immunoglo~ulin solution is dia-lysed for 48 hours against 9 g/l NaCl at 4C. The immunoglo-bulin content is then determined by the A126ocml A128Com method.
Sheep were immunized with a diluted solution of this human immunoglobulin solution according to the following scheme:
day 1 0.5 mg human immunoglobulin dissolved in 0.5 ml physio-logical saline, to which 0.5 ml complete Freunds adju-vant has been added.
Injection: intramuscular.
day 14 1.0 mg human immunoglobulin dissolved in 0.5 ml physio-lGgical saline, to which 0.5 ml incomplete Freunds ad-juvant has been added.
Injection: intramuscular.
day 28 2.0 mg human immunoglobulin dissolved in 0.5 ml physio-logical saline, to which incomplete Freunds adjuvant has been added.
Injection: intramuscular.
day 42 1.0 mg human immunoglobulin dissolved in 1.0 ml physio-logical saline.
Injection: intravenous.
day 56 Sampling of blood.
The immunoglobulin fractions were obtained from the sheep anti-human immunoglobulin sera by salting out according to the above-described 140 g/l Na2S04 method. After washing for the third time, the residue was dissolved in 9 g/l NaCl, after which the immunoglobulin solution was dialysed against a 0.3 " -24-1135~83 g/l NaCl solution in distilled water, adjusted to pH 7.0 with 0.2 mol/l K2C03 dissolved in distilled water.
After centrifuging this dialysed sheep antihuman immunoglobulin solution at 160000 N~kg for 15 minutes, the immunoglobulin content was determined by the A16oCm , A18Com method.
5.4. Preparation of the qold-particle sheep anti-human immunoqlobulin conju~ate The gold sol was ~repared by the method described in 1.1 and the conjugate was prepared in a way analogous to the method described in l.r. whereby in this case the sheep immuno-globulin solution prepared in 5.3. was used, with an immuno-globulin content of 125~ g per ml.
5.5. Test ~rotocol ~ he sample of human sera is diluted 1:25 with 0.2 mol/l Trometamol/HCl/NaCl buffer, pH 7.4, to which 0.5 g/l Tween 20 has been added.
1. 100~ 1 of diluted human serum is pipetted into each well and the whole is incubated for 30 minutes at room temperature.
2. me well is emptied by suction and washed three times with 300~ 1 0.2 mol/l Trometamol/HCl/NaCl buffer, pH 7.4, to which 0.5 g/l Tween 20 has been added.
3. 100~ 1 gold particle sheep anti-human immunoglobulin conju-gate (A536 nm = 1.00) is now pipetted into the well and the whole i4 incubated for 16 hours at room temperature.
4. The well is emptied by suction and washed 3 times with 300,~1 0.01 mol/l Trometamol/HCl/NaCl buffer, pH 7.4, to which 0.5 g/l Tween 20 has been added.
5. Pipette 100~ 1 0.1 mol/l HCl dissolved in distilled water into the well and allow this to act for 30 minutes while shaking.
6. ~fter this, measure the light absorption at 536 nm, using ~1135i83 a small volume spectrophotometer.
5.6. Results An A136Cmnm of about 0.230 was measured for sera with an HAI titre of 512, while a reasonable linear correlation was further found between the HAI titres of the human sera inves-tigated and the A136C nm ~ values obtained in the above-mentioned test.
EXAMPLE VI - Determination of HCG with the aid of an iron oxide -anti-~CG conjugate. ~
6.1. Preparation of the iron oxide sol A solution consisting of 9 mmol/l FeC13 and 1 mmol/l HCl is heated to a temperature of 95C during 65 hours. The initially slightly yellow solution discolours to orange-red due to the formation of iron oxide sol particles. The iron oxide sol obtained in this way consists of cubic iron oxide particles with an edge between ~0 and 80 nm as verified by electron microscopy.
A local light-absorption maximum is found at 370 nm. A sol with aniron oxide content of 0.45 mmol/l exhibits an A137comnm =
1.0 and a pH of 2.7 just after the preparation.
6 2 PreParation of the iron oxide anti-HCG coniuaate Rabbit anti-HCG sera were prepared according to a method analogous to that described for anti HPL sera in example 1.2.
The immunoglobulin fraction hereof was obtained by adding Na2S04, separating the residue, redispersing the latter in a 180 g/l Na2S04 solution and dialysing this dispersion against a solution containing 5 mmol/l NaCl. The immunoglobulin content was determined as described in Example l.3.
The iron oxide sol of 5.1 was diluted, resulting in 30 an iron oxide content o~ 0.4S mmol/l. To 25 ml hereof, 0.5 ml ylutaraldehyde 250 g/l was added and the mixture was incubated ~35~3 during 30 minutes at room temperature and pH 2.8. Subsequent-ly, 0.63 ml rabbit anti-HCG immunoglobulin solution, containing 640 g/ml immunoglobulin, was added.
After an incubation of 1 hour at room temperature and pH 2.7 the pH of the mixture was adjusted to 7.0 with a NaOH solution and the incubation of the mixture was continued overnight at 0-4C.
Before use, the conjugates were washed by centrifug-ing, sucking off the supernatant and resuspending in a buffer 10 consisting of 0.1 mol/l 'rris/NaCl/HCl ~pH 7.4), 1 g/l BS~ and 0.2 g/l Carbowax 20M. Finally, the volume of the sol was ad-justed, so that A1370Cmnm - 1Ø
6.3. Coatinq of Microelisa(R) plates with rabbit anti-HCG
immunoalobulins Microelisa(R) plates were coated with rabbit anti-HCG
immunoglobulin as described for coating with anti-HPL immuno-globulins in example 1.5.
6.4. Test ~rotocol for NCG determination with iron oxide anti-HCG coniuqates A dose response curve was made for HCG, that is solved in a mixture of 0.04 mol/l phosphate buffer, 1 g/l BSA
and 9 g/l NaCl (pH - 7.4), according to the following test protocol.
1. Pipette 0.1 ml of the standard HCG solution into a well of a Microelisa( ) plate, coated with anti-HCG and incubate for 2.5 hours at 37C.
2. Suck the well empty.
3. Pipette 0.1 ml iron oxide anti-HCG (A370cmnm ~ 1.0) into the well and incubate during the night at 37C.
4. Empty the well by suction and wash it 8 times with 0.3 ml of a solution cDtaining 0.1 mol/l l'ris/HCl/~aCl buffer (pH - 7.4) .~.
113~ ?3 and 0.5 g/l Tween 20.
5. Pipette 0.1 ml of a solution containing 0.1 rnol/l NaOH and allow this to act for 15 minutes.
6. Measure the light absorption at 360 nm with a small volume spectrophotometer.
The results of a dose response curve are given in the table below.
HCG concentration in the standard Al cm solution, in mlU/ml -102000 0.367 1000 0.429 500 0.414 250 0.
125 0.275 63 0.205 31 0.179 16 0.172 8 0.154 4 0.142 20 0 0.096 6.5. Determination of HCG in the urine of preqnant women The urines of pregnant women were diluted such that the HCG level lay in the range 1-500 mIU ~ICG/ml. With the aid of the test protocol of 5.4. the HCG concentration of the di-luted samples were determined u~ing the standard curve construct-ed.
EXAMPLE VII - Determination of HCG with the aid of gold particle anti-HCG conjugate in an agglutination test.
The results of the determination of a standard curve are given in the table below.
20 HPL concentration in the standard _53~, ^t t~L~5G
olution, na/ml the test 0 0.030 0.2 0.030 0.8 0.060 3.2 0.106 12.5 0.136 0.188 200 0.220 The solvent for the HPL standard solution is 0.04 mol/l phos-phate buffer + 1 g/l BSA + 9 g/l NaCl, pH 7.4.
1.6.b. Determination of HPL in the serum of pre~nant women.
The sera of pregnant women were diluted such that the HPL level ranged from 5 to 80 ng/ml.
'me diluent for the sera was 0.04 mol/l phosphate buffer, pH 7.4 ~ 1 g/l BSA + 9 g/l NaCl.
With the aid of the test protocol described under 1.6.a., the HPL concentrations of the diluted sera were deter-mined using the standard curve constructed. After correction for the dilution factor, the following results were obtained.
Serum HPL content, EIA test HPL content, MIA
A 4.5 + 0.5 ~g~ ml 4 - 1 ~g~ ml B 3.2 - 0.4 ~g/mll 3.5 - 0.8 ~g/ml C 0.7 + 0.1 ~g/ml 1 + 0.3 ~g/ml EXAMPLE II - Determination of HPL with the aid of gold-particle-labelled antibodies and atomic absorption spectrophotometry.
2.1. Pre~aration of reaqents and coated Microelisa plates The method of preparation of the gold particle rabbit anti-HPL immunoglobulin conjugate and the Microelisa(R) plates coated with rabbit anti-HPL immunoglobulin was the same as des-cribed in Example I.
2.2. Test protocol 2.2.a Determination of standard curve for HPL
In order to construct a standard curve for HPL, ~erial dilutions of HPL were made of consecutively 1, 10, 100 and 1000 ng/ml and a blank. me solvent for HPL was 0.04 mol/l phosphate buffer, pH 7.4, to which 1 g/l BSA and 9 g/l NaCl had been added.
1. Pipette 0.1 ml standard HPL solution into the well of the coated Microelisa plate and incubate for 2 hours at room tem-perature.
2. Suck the well empty and wash with 0.1 ml 0.02 mol/l Tris/HCl buffer, pH 7,~, .~! -15-1~3S1~33 3. Pipette 0.1 ml of the bold particle anti-HPL antibody conju-gate (A136Cmnm = 1.00) into the well and incubate this overnight (about 16~hours~.
4. Suck the well empty and wash it with 0.02 mol/l Tris buffer, pH 7.4.
5. Pipette 0.1 ml HCl, 0.1 mol/l, into the well and allow this to act with shaking on a shaking machine for 30 minutes.
6. ~ow measure the peak value of the light absorption at 242.8 nm in a flame atomic absorption spectrophotometer fitted with a small-volume injector.
A typical example of a standard curve obtained in this way is shown in the table below.
HPL concentration in Peak value of light absorption standard solution at 242.8 nm after termination of the test 0 ng/ml 0.020 1 ng/ml 0.040 10 ng/ml 0.160 100 ng/ml 0.200 201000 ng/ml 0.220 2.2.b. Determination of HPL in the serum of preqnant women.
The sera of pregnant women were diluted such that the HPL level lay in the range 5 - 80 ng/ml. The diluen-t for the sera was 0.04 mol/l phosphate buffer (pH = 7.4) + 1 g/1 BSA
+ 9 g/l NaCl. With the aid of the test protocol described under 2.2.a., the HPL concentration~ of the diluted sera were deter-mined using the standard curve constructed.
EXAMPLE III - Visual detection of hepatitis Bs antigen (HBsAg) by means of gold particle sheep anti-HBs immunoglobulin conju-gate.
3.1. Preparation of the qold sol ' -16-~13~ 3 See example 1.1.
3.2. Preparation of sheep anti-HBs sera Sheep were in~ected with purified HBsAg solution.
3.3. Preparation of the sheep anti-H~s immunoqlobulin solution 1.4 g solid Na2SO4 is added to 10 ml sheep anti-HBsAg serum. After all the sodium sulphate has dissolved, the turbid liquid is allowed to stand for 1 hour at room tempera-ture. ~he liquid is then centrifuged at 25000 N/kg for 10 mi-nutes. me supernatant is removed by suction and the residue is redispersed in 10 ml 140 g/l Na2SO4 solution in distilled water. This procedure is repeated twice.
After centrifuging for the last time and removing the supernatant by suction, the pellet is dissolved in 10 ml of 9 g/1 ~aC1 solution in distilled water. mis solution is dialysed against 6 litres of a 0.3 g/l NaCl solution in dis-tilled water, adjusted to a pH of 7.0 with a solution of 0.2 mol/l K2C03. Dialysis takes place for 16 hours at 4C, after which the dialysed liquid is centrifuged for 20 minutes at 160000 N~kg.
The immunoglobulin solution is stored in portions of 1 ml in vials at -20C. The immunoglobulin content is deter-mined by the A16oCmnm, A18Comnm method already described.
3.4. Pre~aration of the qold-particle sheep anti-HBs immuno-alobulin con1~qate.
The preparation of the conjugate is identical with the method of preparation described in example I point 4, with the exception that a diluted solution (also 125 ng immunoglo-bulin per ml) of the sheep anti-HBs immunoglobulin solution was used instead of the rabbit anti-HPL immunoglobulin solution.
3.5. Coatina of Microelisa( ) plates with sheeP anti-HBs mmunoalobulins : .. - .....
~35~
The coating of Microelisa( ) plates with sheep anti-HBs immunoglobulins is performed in the way described in example I point 5, whereby the sheep anti-HBs immunoglobulin solution was used instead of a rabbit anti-HPL immunoglobulin solution. Ten control wells were included in each plate. These wells were coated with an immunoglobulin solution from human serum which was negative in the following tests:
- Hepanosticon and Hepanostika - Monosticon - Rheumanosticon - Immunodiffusion against normal sheep serum - EIA for anti-HBs.
3.6. Test protocol for a visually readable test.
1. Pipette 0.1 ml of the sample into a well of the coated Micro-elisa plate and incubate for 2 hours at 37C.
2. Wash each well 3x with 0.3 ml 0.02 mol/l Tris/HCl buffer, pH 7.4.
3. Pipette 0.1 ml gold-particle sheep-anti-HBs conjugate 1A136m = 1.00) into the well and incubate overnight at room temperature.
~. Empty the well by suction and wash it 3x with 0.3 ml 0.01 mol/l Tris/HCl/NaCl buffer, pH 7.4.
5. Pipette 0.1 ml 0.1 mol/l HCl solution in distilled water into the well and allow this to act for 30 minutes, on a shaker.
6. A3~ess visually the colour of the liquid in the well and compare it with the control wells present.
Sera which gave a strong positive reaction in the Hepanostika test could generally still be distinguished from the controls in 8-fold dilution.
EXAMPLE IV - Determination of testosterone with the aid of a silver particle testosterone~ -succinyl-bovine serum albumin ~L~351~3 conjugate.
4.1._Preparation of the silver sol ~ .5 ml of a 10 g/l AgNO3 solution in distilled water is diluted with 486~5 ml distilled water and 5.0 ml of a 10 g/l solution of trisodium citrate.2H20 in distilled water is then added. 40.0 ml of a 10 g/l solution of hydrazine in distilled water is added to this solution at room temperature with vigorous stirring using a magnetic stirrer. A grey-yellow-green silver sol with a strong Tyndall scattering is formed within 60 seconds.
After 10-fold dilution with distilled water, the pH was ~.85 and the sol had a light absorption of A1416Cmnm = 0.83.
20 ml of the undiluted sol was centrifuged for 10 minutes at 25000 ~/kg. 15 ml of the supernatant was removed by suction and replaced by 15 ml of a 0.1 g/l trisodium citrate.
2H20 solution in distilled water (pH 6.88).
The washing process was repeated twice. After the final washing, the volume was made up to 20 ml with the 0.1 g/l sodium citrate solution.
After 4-fold dilution with distilled water, the sol 0 had the following properties: `
max = 416 nm 416 nm 1.12 pH = 7.0 4.~. Preparation of testosterone~ -hemisuccinate-BSA
40 mg testosterone-ll ~-hemisuccinate is dissolved in 2 ml dimethyl-formamide and the solution is cooled to -15C.
140 mg bovine serum albumin is dissolved in 3 ml distilled water, after which 1 drop 4N NaOH and 2 ml DMF are added, and the whole is cooled to -15C.
12.5 ~1 N-methylmorpholine and 12.5~11 isobutyl chloroformate are now added to the steroid solution. After 3 1135~33 minutes, the solution is added to the BSA solution. After stir-ring,for 1 hour at -15C and 3 hours at 0C, the solution is transferred to a dialysis bag and dialysed overnight against running tap water. The dialysate is passed through a Sephadex G25TM column, optionally after centrifugation, and the protein fraction is collected and freeze-dried.
4.3. Preparation of antisera for testosterone Rabbits were immunized by injection of 1.25 mg tes-tosterone-ll ~-hemisuccinyl-BSA, dissolved in 2.5 ml physio-logical saline, and 2.5 ml complete Freunds adjuvant (1 ml i.m. 3x, interval 1 week). They were then given an i.v. in-jection of 1 ml of a solution of 1.25 mg testosterone-ll~ -hemi-~uccinyl-BSA in 5 ml physiological saline.
This injection scheme was repeated until the titre of the antiserum was sufficiently high. The rabbits were then bled out, and the sera were stored at -20C until use.
4.4. Preparation of polystyrene tubes coated with rabbit immuno-~lobulin aqainst testosterone A rabbit anti-testosterone immunoglobulin solution is made by salting out with 180 g/l Na2S04 as described in example I point 2.
The dialysed immunoglobulin solution is diluted (to a concentration of 1~ g immunoglobulin/ml) with a buffer con-sisting of 0.1 mol/litre NaH2P04.H20, 60 g/l sucrose, adjusted to pH 7.4 with 4 mol/l NaOH. 1 ml of the immunoglobulin solu-tion i9 pipetted into each 3 ml polystyrene tube, and incubated overnight at room temperature. The tubes are then emptied by suction and filled with 1 ml 10 g/l BSA solution in the above-noted buffer. After two hours, the tubes are emptied by suc-tion and washed 3x with 3 ml distilled water. The tubes are dried completely by suction and are further drled over silica ~L1351S~3 gel overnight. They are then packed in aluminum foil in sets of 25 with a bag of silica gel and stored at 4C.
4.5. Preparation of silver particle-testosterone-ll ~-hemi-succinyl-BSA conjuqate 20 ml of the silver sol, prepared as described in 4.1., was diluted with 80 ml 0.3 g/l NaCl and the pH was adjusted to 7.0 with 0.01 mol/litre NaOH solution in distilled water. A421C nm = 1.08.
3 ml of a solution of testosterone-ll ~-hemisuccinyl-10 BSA, 175 ~ g/ml in 0.3 g/l NaCl adjusted to pH 7.0 with 0.01 mol/l NaOH, is then added dropwise to 15 ml of this sol with vigorous stirring by a magnetic stirrer.
The whole is incubated for about 10 minutes, afterwhich 2 ml 10 g/l Carbowax 20M solution in 0.3 g/l NaCl, adjusted to pH 7.0 with 0.01 mol/l NaOH, is added. The crude conjugate is subsequently washed by centrifuging 10 ml at 25000 N/kg for 20 minutes. 9 ml supernatant is now removed by suction and re-placed by 9 ml washing liquid consisting of 1 g/l Carbowax 20M
solution in 0.3 g/l ~aCl, adjusted to pH 7.0 with 0.01 mol/litre NaOH dissolved in distilled water. After washing 3 times, the conjugate is again centrifuged after which the supernatant liquid is removed by suction. me residue is redispersed in 0.02 mol/litre phosphate buffer, pH 7.4. me conjugate solution now has a pH of 7.4 and A421C nm = 1.08. The washed conjugates are stored at 4C until use.
4.6. Test protocol for the determination of testosterone with the silver particle testosterone-ll ~-hemisuccinyl-BSA coniuqate 1. Pipette a 1 ml sample into a polystyrene tube coated with rabbit anti-testosterone immunoglobulin and incubate for 2 hours at room temperature.
2. Empty the tube by suction and wash 3x with 2 ml 0.02 * - Trademark ~ l 1135~83 mol/litre phosphate buffer, pH 7Ø
3. Pipette 1 ml of the silver particle testosterone~
hemisuccinyl-BSA conju~ate, A42Clmnm = 1.08, into the tube and incubate for 16 hours.
4. Now suck the tube empty and wash with 2 ml 0.02 mol/litre phosphate buffer, p~ 7Ø
5. Pipette 1 ml 0.1 mol/litre HCl, solution in distilled water, into the tubes and allow this to act for 30 minutes.
6. Measure the liquid in the tube A121Cm 4.7. Results Ihe test media were colourimetrically determined with the aid of a standard curve, whereby amounts of testosterone of the magnitude of 0.5 nc3/ml and greater could be determined.
EX~PLE V - Determination of the titre of human anti-Rubella sera 5.1. Preparation of the Rubella anti~en 200 ml of a host cell suspension (BHK 21C3 from the Rijks Instituut voor Volksgezondheid, RIV) in culture medium (concentration 10 live cells per ml) is introduced into a roller bottle (surface 490 CM ). The cell culture is lcept for 16 to 20 hours at 37C. The culture medium is tnen removed and the cell layer is washed twice with 20 ml phosphate-buffered saline solution (PBS). 10 ml ~irus suspension (virus type ~33 RI~) is th~n diluted 1:100 with PBS and introduced into the bottle, after which the whole i~ :incubated at 37C for about 2 1/2 hours.
100 ml maintenance medium is now added and the cul-ture is then kept at 37C. After a period of at least 64 hours, but not exceeding 136 hours, depending on the assessment of the cytopathic effecl, the incubatecl mono]ayer of infected cells is harbe.sted. The mairltenarlce mediurrl is remove(l, and the mono-, . ...
11351~33 layer of cells is washed once with 20 ml PBS. The cells are loosened from the wall by means of sterile glass beads, taken up in 90 ml PBS and frozen at -20C.
After thawing, 10 ml glycine solution (1 mol/l), the pH being adjusted to 9.0 with solid NaOH, is added to 90 ml cell suspension, and the whole is thoroughly mixed for 6 hours at 37C. The cell suspension is now ultrascnicated for 30 minutes, with cooling in ice, and subsequently centrifuged at 30000 ~kg for 30 minutes at 4C. The supernatant containing the Rubella virus is collected and inactivated by consecutive addition of:
1:100 (v/v)NaOH (1 mol/litre) solution in distilled water 1:100 (v/v) freshly prepared 10%~ -propiolactone solution in distilled water, The liquid is allowed to stand for 48 hours at 4C, after which it is centrifuged for 30 minutes at 30000 N~kg and 4C.
me supernatant is the liquid containing the Rubella virus, which can if necessary be stored at -20C until use.
5.2. Coatinq of Microelisa( ) plates with Rubella antiaens.
The Rubella antigen solution, prepared as described in 5.1., is diluted 1:100 with a 0.05 mol/litre carbonate buffer, pH 9.6. 100 ~1 of this liquid Rubella antigen solution is pipetted into each well of the Microelisa plate and the whole is subsequently incubated for 16 hours at 4C. The well of the Microelisa(R) plate are now emptied by suction and washed 3 times with 300 ~1 0.05 mol/l phosphate buffer, pH 7.4, to which 9 g/l NaC1 and 0.5 g/l Tween 20 have been added.
5.3. Preparation of sheet anti-human immuno~lobulin solution Normal human serum is salted out with 140 g/1 Na2SO4, which i5 added as a solid in small portions. After standing for 1 hour at room temperature, the turbid liquid is centri-1~351~3 fuged at 25000 N~kg for 10 minutes. The supernatant is removed by suction, after which the residue is washed with a 140 g/l Na2S04 solution in distilled water. After centrifuging and removal of the supernatant by suction, the residue is dissolved in 9 g/l ~aCl, after which the immunoglo~ulin solution is dia-lysed for 48 hours against 9 g/l NaCl at 4C. The immunoglo-bulin content is then determined by the A126ocml A128Com method.
Sheep were immunized with a diluted solution of this human immunoglobulin solution according to the following scheme:
day 1 0.5 mg human immunoglobulin dissolved in 0.5 ml physio-logical saline, to which 0.5 ml complete Freunds adju-vant has been added.
Injection: intramuscular.
day 14 1.0 mg human immunoglobulin dissolved in 0.5 ml physio-lGgical saline, to which 0.5 ml incomplete Freunds ad-juvant has been added.
Injection: intramuscular.
day 28 2.0 mg human immunoglobulin dissolved in 0.5 ml physio-logical saline, to which incomplete Freunds adjuvant has been added.
Injection: intramuscular.
day 42 1.0 mg human immunoglobulin dissolved in 1.0 ml physio-logical saline.
Injection: intravenous.
day 56 Sampling of blood.
The immunoglobulin fractions were obtained from the sheep anti-human immunoglobulin sera by salting out according to the above-described 140 g/l Na2S04 method. After washing for the third time, the residue was dissolved in 9 g/l NaCl, after which the immunoglobulin solution was dialysed against a 0.3 " -24-1135~83 g/l NaCl solution in distilled water, adjusted to pH 7.0 with 0.2 mol/l K2C03 dissolved in distilled water.
After centrifuging this dialysed sheep antihuman immunoglobulin solution at 160000 N~kg for 15 minutes, the immunoglobulin content was determined by the A16oCm , A18Com method.
5.4. Preparation of the qold-particle sheep anti-human immunoqlobulin conju~ate The gold sol was ~repared by the method described in 1.1 and the conjugate was prepared in a way analogous to the method described in l.r. whereby in this case the sheep immuno-globulin solution prepared in 5.3. was used, with an immuno-globulin content of 125~ g per ml.
5.5. Test ~rotocol ~ he sample of human sera is diluted 1:25 with 0.2 mol/l Trometamol/HCl/NaCl buffer, pH 7.4, to which 0.5 g/l Tween 20 has been added.
1. 100~ 1 of diluted human serum is pipetted into each well and the whole is incubated for 30 minutes at room temperature.
2. me well is emptied by suction and washed three times with 300~ 1 0.2 mol/l Trometamol/HCl/NaCl buffer, pH 7.4, to which 0.5 g/l Tween 20 has been added.
3. 100~ 1 gold particle sheep anti-human immunoglobulin conju-gate (A536 nm = 1.00) is now pipetted into the well and the whole i4 incubated for 16 hours at room temperature.
4. The well is emptied by suction and washed 3 times with 300,~1 0.01 mol/l Trometamol/HCl/NaCl buffer, pH 7.4, to which 0.5 g/l Tween 20 has been added.
5. Pipette 100~ 1 0.1 mol/l HCl dissolved in distilled water into the well and allow this to act for 30 minutes while shaking.
6. ~fter this, measure the light absorption at 536 nm, using ~1135i83 a small volume spectrophotometer.
5.6. Results An A136Cmnm of about 0.230 was measured for sera with an HAI titre of 512, while a reasonable linear correlation was further found between the HAI titres of the human sera inves-tigated and the A136C nm ~ values obtained in the above-mentioned test.
EXAMPLE VI - Determination of HCG with the aid of an iron oxide -anti-~CG conjugate. ~
6.1. Preparation of the iron oxide sol A solution consisting of 9 mmol/l FeC13 and 1 mmol/l HCl is heated to a temperature of 95C during 65 hours. The initially slightly yellow solution discolours to orange-red due to the formation of iron oxide sol particles. The iron oxide sol obtained in this way consists of cubic iron oxide particles with an edge between ~0 and 80 nm as verified by electron microscopy.
A local light-absorption maximum is found at 370 nm. A sol with aniron oxide content of 0.45 mmol/l exhibits an A137comnm =
1.0 and a pH of 2.7 just after the preparation.
6 2 PreParation of the iron oxide anti-HCG coniuaate Rabbit anti-HCG sera were prepared according to a method analogous to that described for anti HPL sera in example 1.2.
The immunoglobulin fraction hereof was obtained by adding Na2S04, separating the residue, redispersing the latter in a 180 g/l Na2S04 solution and dialysing this dispersion against a solution containing 5 mmol/l NaCl. The immunoglobulin content was determined as described in Example l.3.
The iron oxide sol of 5.1 was diluted, resulting in 30 an iron oxide content o~ 0.4S mmol/l. To 25 ml hereof, 0.5 ml ylutaraldehyde 250 g/l was added and the mixture was incubated ~35~3 during 30 minutes at room temperature and pH 2.8. Subsequent-ly, 0.63 ml rabbit anti-HCG immunoglobulin solution, containing 640 g/ml immunoglobulin, was added.
After an incubation of 1 hour at room temperature and pH 2.7 the pH of the mixture was adjusted to 7.0 with a NaOH solution and the incubation of the mixture was continued overnight at 0-4C.
Before use, the conjugates were washed by centrifug-ing, sucking off the supernatant and resuspending in a buffer 10 consisting of 0.1 mol/l 'rris/NaCl/HCl ~pH 7.4), 1 g/l BS~ and 0.2 g/l Carbowax 20M. Finally, the volume of the sol was ad-justed, so that A1370Cmnm - 1Ø
6.3. Coatinq of Microelisa(R) plates with rabbit anti-HCG
immunoalobulins Microelisa(R) plates were coated with rabbit anti-HCG
immunoglobulin as described for coating with anti-HPL immuno-globulins in example 1.5.
6.4. Test ~rotocol for NCG determination with iron oxide anti-HCG coniuqates A dose response curve was made for HCG, that is solved in a mixture of 0.04 mol/l phosphate buffer, 1 g/l BSA
and 9 g/l NaCl (pH - 7.4), according to the following test protocol.
1. Pipette 0.1 ml of the standard HCG solution into a well of a Microelisa( ) plate, coated with anti-HCG and incubate for 2.5 hours at 37C.
2. Suck the well empty.
3. Pipette 0.1 ml iron oxide anti-HCG (A370cmnm ~ 1.0) into the well and incubate during the night at 37C.
4. Empty the well by suction and wash it 8 times with 0.3 ml of a solution cDtaining 0.1 mol/l l'ris/HCl/~aCl buffer (pH - 7.4) .~.
113~ ?3 and 0.5 g/l Tween 20.
5. Pipette 0.1 ml of a solution containing 0.1 rnol/l NaOH and allow this to act for 15 minutes.
6. Measure the light absorption at 360 nm with a small volume spectrophotometer.
The results of a dose response curve are given in the table below.
HCG concentration in the standard Al cm solution, in mlU/ml -102000 0.367 1000 0.429 500 0.414 250 0.
125 0.275 63 0.205 31 0.179 16 0.172 8 0.154 4 0.142 20 0 0.096 6.5. Determination of HCG in the urine of preqnant women The urines of pregnant women were diluted such that the HCG level lay in the range 1-500 mIU ~ICG/ml. With the aid of the test protocol of 5.4. the HCG concentration of the di-luted samples were determined u~ing the standard curve construct-ed.
EXAMPLE VII - Determination of HCG with the aid of gold particle anti-HCG conjugate in an agglutination test.
7.1. Preparation of reaqents A gold sol is prepared according to the method des-cribed in 1.1. and rabbit anti-HCG sera and immunoglobulin 1~351~3 fractions hereof as described in 5.2.
A gold particle anti-HCG conjugate is prepared with the gold sol and the rabbit anti-HCG solutions according to the method described in 1.4.
7.2. Test ~rotocol 1. Pipette 1 ml of the gold particle anti-HCG conjugate (A540Cm m = 1.50) into a test tube of approximately 3 ml.
2. Pipette herein 0.1 ml of the urine to be tested and mix.
3. Judge the colour of the conjugate after an incubation of 1 hour at room temperature:
- a colour change from red to colourless indicates that the sample is strongly positive - a colour change from red to purple indicates that the sample is positive and contains 1 IU HCG/ml or more - if the colour does not change the sample is negative, i.e. it contains 0.4 IU HCG/ml or less.
7.3. Results With the aid of the test protocol of 7.2. it is pos-sible to diagnose pregnancy on the 33-35th day after the last menstruation.
EXAMPLE VIII - Competitive Receptor assay for HCG
A gold particle anti-HCG conjugate is prepared with the gold sol and the rabbit anti-HCG solutions according to the method described in 1.4.
7.2. Test ~rotocol 1. Pipette 1 ml of the gold particle anti-HCG conjugate (A540Cm m = 1.50) into a test tube of approximately 3 ml.
2. Pipette herein 0.1 ml of the urine to be tested and mix.
3. Judge the colour of the conjugate after an incubation of 1 hour at room temperature:
- a colour change from red to colourless indicates that the sample is strongly positive - a colour change from red to purple indicates that the sample is positive and contains 1 IU HCG/ml or more - if the colour does not change the sample is negative, i.e. it contains 0.4 IU HCG/ml or less.
7.3. Results With the aid of the test protocol of 7.2. it is pos-sible to diagnose pregnancy on the 33-35th day after the last menstruation.
EXAMPLE VIII - Competitive Receptor assay for HCG
8.1. Preparation of purified HCG receptor protein from bovine corPora lutea (BCL) HCG receptor protein from BCL was prepared by the method described in detail by Khan and Saxena in Enzyme Labelled Irnmunoassay of Hormones and Drugs ed. S.B. Pal, Publ.
Walter de Gruyter Berlin New York 1978. Fresh BCL tissue was pulverized, homogenized, filtered, further homogenized and centrifuged at 6500 N~kg. The supernate was centrifuged at 30 130,000 N/kg, the obtained pellct hornogenized and centrifuged in a zonal rotor with a sucrose gradient and elllted. q~he Ereeze ~135~183 dried fractions containing the HCG receptor membranes were re-dissolved and after centrifugation Mannitol was added to the clear supernate, whereafter the pH was adjusted to 7Ø The ob-tained dispersion was purified by affinity chromatograph on CNBr-activated sepharose 4B coupled with Human LH. After elution of the bound receptor, the pH was immediately adjusted to 7 and Triton X-100 was removed by adsorption on Bio-beads. The obtain-ed dispersion of HCG receptor protein was thoroughly dialyzed against PBS, pH 7.0 and stored at 4C.
8.2. Coatinq of Polvstyrene micro titration plates with HCG
receptor protein To this end a HCG receptor protein receptor solution is prepared with a content of 25 ~g protein/ml by diluting the HCG receptor protein in PBS with a solution of 0.04 mol/l ~a2HPO4, adjusted to a pH of 7.4 with a solution of 0.04 mol/l NaH2PO4 to which 0.1 g/l Merthiolate is added. Micro titration plates were coated with this HCG receptor solution according to the protocol described in 1.5.
8.3. Preparation of a qold dispersion A gold dispersion consisting of particles having a diameter between 6 and 15 nm was prepared by adding 14 ml of a solution containing lO g/l sodium citrate 2H2O to a boiling solution containing lO0 mg/l chloro-auric acid (HAUCl4), and the mixture is kept boiling for 15 minutes. The obtained orange red gold dispersion is cooled down to room temperature and re-adjusted to a volume of 500 ml in a volumetric flask. The gold dispersion had a light absorption maximum at 523 nm and A52C3mnm = 1Ø
8.4. Preparation of a ~old Particle HCG coniuqate A solution containing 5 mg purified HCG/ml in PBS
(p~ = 7.0) was thorouyhly dialyzed against a solution contain-~13S1~3 ing 5 mol/l NaCl, adjusted to a pH of 4.5 by means of HCl.
1 ml of the dialyzed HCG solution was added to 500 ml of the gold dispersion and stirred for 10 minutes at room temperature.
10 ml of a solution containing 10 g Carbowax 20 M/l was added to the fresh conjugate and incubated for another 10 minutes. There-after the pH was adjusted to 7.4 by means of a K2C03 solution.
The conjugate was stored at 4C. Just before use 1 ml of a 1 mol/l TRIS/HCl/~aCl buffer (pH - 7.4) containing 1 g/l BSA
was added to 10 ml of the stored conjugate dispersion.
8.5. Test protocol for a competitive HCG assay 100 ~1 of a standard HCG solution (or the sample) was added to a well of micro titration plates coated with HCG
receptor protein and incubated for one hour at room temperature.
Thereafter 100 ~1 of the buffered gold particle HCG conjugate was added and incubated for four hours at room te~perature. The well was emptied and washed with 0.3 ml 0.01 mol/l TRIS/HCl/~aCl buffer pH = 7.4.
Finally 0.1 ml 0.1 mol/l NaOH solution in water was added and incubated for 10 minutes. The light absorption of the fluid in a well was measured at 536 nm using a small volume ~pectrophotometer.
8.6. Result A dose-response curve for free HCG was obtained in a HCG concentration range between O and 1000 mIU/ml having a mid-point near 100 mIU }ICG/ml. q'he detection limit was about 10 mIU HCG/ml. The IICG concentration measured in samples was usually higher than determined with an EIA.
EXAMPLE ~X - Sandwich assay for HCG using an insolubilized HCG
receptor and a gold particle-anti HCG conjugate For this assay microtitration plates were coated with HCG receptor protein as describcd in Example ~.2. A yold par-~1351~3 ticle-anti HCG conjugate was prepared as described in Example 1.4. using rabbit anti-HCG instead of rabbit anti-HPL.
Walter de Gruyter Berlin New York 1978. Fresh BCL tissue was pulverized, homogenized, filtered, further homogenized and centrifuged at 6500 N~kg. The supernate was centrifuged at 30 130,000 N/kg, the obtained pellct hornogenized and centrifuged in a zonal rotor with a sucrose gradient and elllted. q~he Ereeze ~135~183 dried fractions containing the HCG receptor membranes were re-dissolved and after centrifugation Mannitol was added to the clear supernate, whereafter the pH was adjusted to 7Ø The ob-tained dispersion was purified by affinity chromatograph on CNBr-activated sepharose 4B coupled with Human LH. After elution of the bound receptor, the pH was immediately adjusted to 7 and Triton X-100 was removed by adsorption on Bio-beads. The obtain-ed dispersion of HCG receptor protein was thoroughly dialyzed against PBS, pH 7.0 and stored at 4C.
8.2. Coatinq of Polvstyrene micro titration plates with HCG
receptor protein To this end a HCG receptor protein receptor solution is prepared with a content of 25 ~g protein/ml by diluting the HCG receptor protein in PBS with a solution of 0.04 mol/l ~a2HPO4, adjusted to a pH of 7.4 with a solution of 0.04 mol/l NaH2PO4 to which 0.1 g/l Merthiolate is added. Micro titration plates were coated with this HCG receptor solution according to the protocol described in 1.5.
8.3. Preparation of a qold dispersion A gold dispersion consisting of particles having a diameter between 6 and 15 nm was prepared by adding 14 ml of a solution containing lO g/l sodium citrate 2H2O to a boiling solution containing lO0 mg/l chloro-auric acid (HAUCl4), and the mixture is kept boiling for 15 minutes. The obtained orange red gold dispersion is cooled down to room temperature and re-adjusted to a volume of 500 ml in a volumetric flask. The gold dispersion had a light absorption maximum at 523 nm and A52C3mnm = 1Ø
8.4. Preparation of a ~old Particle HCG coniuqate A solution containing 5 mg purified HCG/ml in PBS
(p~ = 7.0) was thorouyhly dialyzed against a solution contain-~13S1~3 ing 5 mol/l NaCl, adjusted to a pH of 4.5 by means of HCl.
1 ml of the dialyzed HCG solution was added to 500 ml of the gold dispersion and stirred for 10 minutes at room temperature.
10 ml of a solution containing 10 g Carbowax 20 M/l was added to the fresh conjugate and incubated for another 10 minutes. There-after the pH was adjusted to 7.4 by means of a K2C03 solution.
The conjugate was stored at 4C. Just before use 1 ml of a 1 mol/l TRIS/HCl/~aCl buffer (pH - 7.4) containing 1 g/l BSA
was added to 10 ml of the stored conjugate dispersion.
8.5. Test protocol for a competitive HCG assay 100 ~1 of a standard HCG solution (or the sample) was added to a well of micro titration plates coated with HCG
receptor protein and incubated for one hour at room temperature.
Thereafter 100 ~1 of the buffered gold particle HCG conjugate was added and incubated for four hours at room te~perature. The well was emptied and washed with 0.3 ml 0.01 mol/l TRIS/HCl/~aCl buffer pH = 7.4.
Finally 0.1 ml 0.1 mol/l NaOH solution in water was added and incubated for 10 minutes. The light absorption of the fluid in a well was measured at 536 nm using a small volume ~pectrophotometer.
8.6. Result A dose-response curve for free HCG was obtained in a HCG concentration range between O and 1000 mIU/ml having a mid-point near 100 mIU }ICG/ml. q'he detection limit was about 10 mIU HCG/ml. The IICG concentration measured in samples was usually higher than determined with an EIA.
EXAMPLE ~X - Sandwich assay for HCG using an insolubilized HCG
receptor and a gold particle-anti HCG conjugate For this assay microtitration plates were coated with HCG receptor protein as describcd in Example ~.2. A yold par-~1351~3 ticle-anti HCG conjugate was prepared as described in Example 1.4. using rabbit anti-HCG instead of rabbit anti-HPL.
9.1. Test Protocol 0.1 ml of a standard solution HCG in 0.1 mol/l TRIS/HCl/NaCl buffer pH = 7.4 containing 1 g/l BSA (or sample) is pipetted into a well of the microtitration plate coated with HCG receptor protein and incubated for 1 hour at room temperature.
men the wells are emptied and 0.1 ml of the gold particle anti HCG conjugate is pipetted into each well and incubated over-night at room temperature. The wells are emptied and washed 6times with 0.3 ml 0.01 mol/1 TRIS/HCl/NaCl buffer pH = 7.4.
0.1 ml and 0.1 mol/l NaOH is pipetted into each well and incubated for 15 minutes. me light absorption at 536 nm of the content of the wells is measured using a small volume spectrophotometer.
9 2 Test results Dose response curves were obtained for HCG concen-tration between 0 and 1000 mIU/ml. The detection limit was about 5 mIU/ml. HCG concentrations measured in samples were about the same as measured with EIA.
....
men the wells are emptied and 0.1 ml of the gold particle anti HCG conjugate is pipetted into each well and incubated over-night at room temperature. The wells are emptied and washed 6times with 0.3 ml 0.01 mol/1 TRIS/HCl/NaCl buffer pH = 7.4.
0.1 ml and 0.1 mol/l NaOH is pipetted into each well and incubated for 15 minutes. me light absorption at 536 nm of the content of the wells is measured using a small volume spectrophotometer.
9 2 Test results Dose response curves were obtained for HCG concen-tration between 0 and 1000 mIU/ml. The detection limit was about 5 mIU/ml. HCG concentrations measured in samples were about the same as measured with EIA.
....
Claims (83)
1. A method for the immunochemical determination of a first component of an immunological reaction system in an aqueous medium, comprising:
a) bringing a sample into contact with 1) a known amount of a reagent selected from the group consisting of metal sol particles which have an immunological component coupled to their surfaces, metal sol particles which have coupled to their surfaces (i) an immunological component, and (ii) a macromolecule which is immunologically inert, and metal sol particles coated with an immunologically inert hydrophilic polymer or copolymer, the surface of each coated particle having coupled to it an immunological component wherein the immuno-logical component of said reagent is similar to said first component and 2) a known amount of an insolubilized second component of said system-capable of reacting immunologically with said first component and said reagent, b) allowing the immunological reaction to take place, whereby a fraction bound to said insolubilized component and a fraction which is free from said insolubilized component are produced, c) and determining the amount of the metal of the metal sol particles in one of said fractions, which is a measure of the amount of said first component in said sample.
a) bringing a sample into contact with 1) a known amount of a reagent selected from the group consisting of metal sol particles which have an immunological component coupled to their surfaces, metal sol particles which have coupled to their surfaces (i) an immunological component, and (ii) a macromolecule which is immunologically inert, and metal sol particles coated with an immunologically inert hydrophilic polymer or copolymer, the surface of each coated particle having coupled to it an immunological component wherein the immuno-logical component of said reagent is similar to said first component and 2) a known amount of an insolubilized second component of said system-capable of reacting immunologically with said first component and said reagent, b) allowing the immunological reaction to take place, whereby a fraction bound to said insolubilized component and a fraction which is free from said insolubilized component are produced, c) and determining the amount of the metal of the metal sol particles in one of said fractions, which is a measure of the amount of said first component in said sample.
2. A method for the immunochemical determination of a first component of an immunological reaction system in an aqueous medium, comprising:
a) bringing a sample into contact with 1) a known amount of a reagent selected from the group consisting of metal sol particles which have an immunological component coupled to their surfaces, metal sol particles which have coupled to their surfaces (i) an immunological component and (ii) a macro-molecule which is immunologically inert, and metal sol particles coated with an immunologically inert hydrophilic polymer or copolymer, the surface of each coated particle having coupled to it an immunological component, wherein the immunological component of said reagent is a second component of said system capable of reacting immunologically with said first component and 2) a known amount of an insolubilized amount of an immuno-logical component which is similar to said first component and capable of reacting with said reagent, b) allowing the immunological reaction to take place whereby a fraction bound to said insolubilized component and a fraction which is free from said insolubilized component are produced, c) and determining the amount of metal of the metal sol particles in one of said fractions, which is a measure of the amount of said first component in said sample.
a) bringing a sample into contact with 1) a known amount of a reagent selected from the group consisting of metal sol particles which have an immunological component coupled to their surfaces, metal sol particles which have coupled to their surfaces (i) an immunological component and (ii) a macro-molecule which is immunologically inert, and metal sol particles coated with an immunologically inert hydrophilic polymer or copolymer, the surface of each coated particle having coupled to it an immunological component, wherein the immunological component of said reagent is a second component of said system capable of reacting immunologically with said first component and 2) a known amount of an insolubilized amount of an immuno-logical component which is similar to said first component and capable of reacting with said reagent, b) allowing the immunological reaction to take place whereby a fraction bound to said insolubilized component and a fraction which is free from said insolubilized component are produced, c) and determining the amount of metal of the metal sol particles in one of said fractions, which is a measure of the amount of said first component in said sample.
3. A method for the immunochemical determination of a first component of an immunological reaction system in an aqueous medium, comprising:
a) binding an amount of a second component of said system capable of reacting immunologically with said first component to the surface of a water-insoluble, water insuspens-ible, solid carrier, b) contacting said bound second component with a sample, c) allowing the immunological reaction to take place, d) contacting said bound second component with a known amount of a reagent selected from the group consisting of metal sol particles which have an immunological component coupled to their surfaces, metal sol particles which have coupled to their surfaces (i) an immunological component, and (ii) a macromolecule which is immunologically inert, and metal sol particles coated with an immunologically inert hydrophilic polymer or copolymer, the surface of each coated particle having coupled to it an immunological component, wherein the immuno-logical component of said reagent is capable of reacting with said first component, to bind said reagent to that part of said bound second component which has undergone the reaction in step (c), e) and determining the amount of the metal of the metal sol particles in the free reagent or the bound reagent, which is a measure of the amount of said first component in said sample.
a) binding an amount of a second component of said system capable of reacting immunologically with said first component to the surface of a water-insoluble, water insuspens-ible, solid carrier, b) contacting said bound second component with a sample, c) allowing the immunological reaction to take place, d) contacting said bound second component with a known amount of a reagent selected from the group consisting of metal sol particles which have an immunological component coupled to their surfaces, metal sol particles which have coupled to their surfaces (i) an immunological component, and (ii) a macromolecule which is immunologically inert, and metal sol particles coated with an immunologically inert hydrophilic polymer or copolymer, the surface of each coated particle having coupled to it an immunological component, wherein the immuno-logical component of said reagent is capable of reacting with said first component, to bind said reagent to that part of said bound second component which has undergone the reaction in step (c), e) and determining the amount of the metal of the metal sol particles in the free reagent or the bound reagent, which is a measure of the amount of said first component in said sample.
4. A method for the immunochemical determination of human placental lactogen (HPL), comprising:
a) binding a known amount of rabbit anti-HPL
immunoglobulin to the surface of a water-insoluble, water-insuspensible, solid carrier, b) contacting said bound immunoglobulin with a sample solution, c) allowing the immunological reaction between the bound immunoglobulin and any HPL in the sample solution to take place, d) washing the solid carrier, e) contacting said bound immunoglobulin with a known amount of a reagent, said reagent consisting essentially of gold sol particles, said particles having rabbit anti-HPL
immunoglobulin coupled to their surfaces, to bind said reagent to that part of said bound immunoglobulin which has undergone the reaction in step (c), f) and determining the amount of gold in the free reagent or the bound reagent, which is a measure of the amount of HPL in said sample solution.
a) binding a known amount of rabbit anti-HPL
immunoglobulin to the surface of a water-insoluble, water-insuspensible, solid carrier, b) contacting said bound immunoglobulin with a sample solution, c) allowing the immunological reaction between the bound immunoglobulin and any HPL in the sample solution to take place, d) washing the solid carrier, e) contacting said bound immunoglobulin with a known amount of a reagent, said reagent consisting essentially of gold sol particles, said particles having rabbit anti-HPL
immunoglobulin coupled to their surfaces, to bind said reagent to that part of said bound immunoglobulin which has undergone the reaction in step (c), f) and determining the amount of gold in the free reagent or the bound reagent, which is a measure of the amount of HPL in said sample solution.
5. A method for the immunochemical determination of hepatitis Bs antigen (HBsAg), comprising:
a) binding a known amount of sheep anti-HBsAg immunoglobulin to the surface of a water-insoluble, water-insuspensible, solid carrier, b) contacting said bound immunoglobulin with a sample solution, c) allowing the immunological reaction between the bound immunoglobulin and any HBsAg in the sample solution to take place, d) washing the solid carrier, e) contacting said bound immunoglobulin with a known amount of a reagent, said reagent consisting essentially of gold sol particles, said particles having anti-HBsAg immunoglobulin coupled to their surfaces, to bind said reagent to that part of said bound immunoglobulin which has undergone the reaction in step (c), f) and determining the amount of gold in the free reagent or the bound reagent, which is a measure of the amount of HBsAg.
a) binding a known amount of sheep anti-HBsAg immunoglobulin to the surface of a water-insoluble, water-insuspensible, solid carrier, b) contacting said bound immunoglobulin with a sample solution, c) allowing the immunological reaction between the bound immunoglobulin and any HBsAg in the sample solution to take place, d) washing the solid carrier, e) contacting said bound immunoglobulin with a known amount of a reagent, said reagent consisting essentially of gold sol particles, said particles having anti-HBsAg immunoglobulin coupled to their surfaces, to bind said reagent to that part of said bound immunoglobulin which has undergone the reaction in step (c), f) and determining the amount of gold in the free reagent or the bound reagent, which is a measure of the amount of HBsAg.
6. A method for the immunochemical determination of testosterone, comprising:
a) binding a known amount of rabbit anti-testosterone immunoglobulin to the surface of a water-insoluble, water-insuspensible, solid carrier, b) contacting said bound immunoglobulin with a sample solution, c) allowing the immunological reaction between the bound immunoglobulin and any testosterone in the sample solu-tion to take place, d) washing the solid carrier, e) after said reaction, contacting said bound immunoglobulin with a known amount of a reagent, said reagent consisting essentially of silver sol particles, said particles having testosterone-ll.alpha.-hemisuccinyl-bovine serum albumin coupled to their surfaces to form a silver particle-testo-sterone-ll.alpha.-hemisuccinyl-bovine serum albumin conjugate, said contacting of said reagent resulting in the binding of said reagent to that part of said bound immunoglobulin which has undergone the reaction in step (c), f) and determining the amount of silver in the free reagent or the bound reagent, which is a measure of the amount of testosterone.
a) binding a known amount of rabbit anti-testosterone immunoglobulin to the surface of a water-insoluble, water-insuspensible, solid carrier, b) contacting said bound immunoglobulin with a sample solution, c) allowing the immunological reaction between the bound immunoglobulin and any testosterone in the sample solu-tion to take place, d) washing the solid carrier, e) after said reaction, contacting said bound immunoglobulin with a known amount of a reagent, said reagent consisting essentially of silver sol particles, said particles having testosterone-ll.alpha.-hemisuccinyl-bovine serum albumin coupled to their surfaces to form a silver particle-testo-sterone-ll.alpha.-hemisuccinyl-bovine serum albumin conjugate, said contacting of said reagent resulting in the binding of said reagent to that part of said bound immunoglobulin which has undergone the reaction in step (c), f) and determining the amount of silver in the free reagent or the bound reagent, which is a measure of the amount of testosterone.
7. A method for the immunochemical determination of the titer of human anti-Rubella sera, comprising:
a) binding a known amount of Rubella antigen to the surface of a water-insoluble, water-insuspensible, solid carrier, b) contacting said bound antigen with a human serum sample, c) allowing the immunological reaction between the bound antigen and the serum to take place, d) washing the solid carrier, e) contacting said bound antigen with a known amount of a reagent, said reagent consisting essentially of gold sol particles, said particles having sheep anti-human immuno-globulin coupled to their surfaces to form a gold particle sheep antihuman immunoglobulin conjugate, said contacting of said reagent resulting in the binding of said reagent to that part of said bound antigen which has undergone the reaction in step (c), f) and determining the amount of gold in the free reagent or the bound reagent, which is a measure of the titer of the human anti-Rubella serum sample.
a) binding a known amount of Rubella antigen to the surface of a water-insoluble, water-insuspensible, solid carrier, b) contacting said bound antigen with a human serum sample, c) allowing the immunological reaction between the bound antigen and the serum to take place, d) washing the solid carrier, e) contacting said bound antigen with a known amount of a reagent, said reagent consisting essentially of gold sol particles, said particles having sheep anti-human immuno-globulin coupled to their surfaces to form a gold particle sheep antihuman immunoglobulin conjugate, said contacting of said reagent resulting in the binding of said reagent to that part of said bound antigen which has undergone the reaction in step (c), f) and determining the amount of gold in the free reagent or the bound reagent, which is a measure of the titer of the human anti-Rubella serum sample.
8. A method for the immunochemical determination of HCG, comprising:
a) binding a known amount of rabbit anti-HCG
immunoglobulin to the surface of a water-insoluble, water-insuspensible, solid carrier, b) contacting said bound immunoglobulin with a sample solution, c) allowing the immunological reaction between the bound immunoglobulin and any HCG in the sample solution to take place, d) washing the solid carrier, e) contacting said bound immunoglobulin with a known amount of reagent, said reagent consisting essentially of iron oxide sol particles, said particles having anti-HCG immuno-globulin coupled to their surfaces, to bind said reagent to that part of said bound immunoglobulin which has undergone the reaction in step (c), f) and determining the amount of iron oxide in the free reagent or the bound reagent which is a measure of the amount of HCG in the sample solution.
a) binding a known amount of rabbit anti-HCG
immunoglobulin to the surface of a water-insoluble, water-insuspensible, solid carrier, b) contacting said bound immunoglobulin with a sample solution, c) allowing the immunological reaction between the bound immunoglobulin and any HCG in the sample solution to take place, d) washing the solid carrier, e) contacting said bound immunoglobulin with a known amount of reagent, said reagent consisting essentially of iron oxide sol particles, said particles having anti-HCG immuno-globulin coupled to their surfaces, to bind said reagent to that part of said bound immunoglobulin which has undergone the reaction in step (c), f) and determining the amount of iron oxide in the free reagent or the bound reagent which is a measure of the amount of HCG in the sample solution.
9. A method for the immunochemical determination of HCG, comprising:
a) binding a known amount of rabbit anti-HCG immuno-globulin to the surface of a water-insoluble, water insuspens-ible, solid carrier, b) contacting said bound immunoglobulin with a sample solution, c) allowing the immunological reaction between the bound immunoglobulin and any HCG in the sample solution to take place, d) washing the solid carrier, e) contacting said bound immunoglobulin with a known amount of reagent, said reagent consisting essentially of gold sol particles, said particles having anti-HCG immunoglobulin coupled to their surfaces, to bind said reagent to that part of said bound immunoglobulin which has undergone the reaction in step (c), f) and determining the amount of gold in the free reagent or the bound reagent which is a measure of the amount of HCG in the sample solution.
a) binding a known amount of rabbit anti-HCG immuno-globulin to the surface of a water-insoluble, water insuspens-ible, solid carrier, b) contacting said bound immunoglobulin with a sample solution, c) allowing the immunological reaction between the bound immunoglobulin and any HCG in the sample solution to take place, d) washing the solid carrier, e) contacting said bound immunoglobulin with a known amount of reagent, said reagent consisting essentially of gold sol particles, said particles having anti-HCG immunoglobulin coupled to their surfaces, to bind said reagent to that part of said bound immunoglobulin which has undergone the reaction in step (c), f) and determining the amount of gold in the free reagent or the bound reagent which is a measure of the amount of HCG in the sample solution.
10. A method for the immunochemical determination of HCG, comprising a) binding a known amount of a HCG receptor protein to the surface of a water insoluble, water insuspensible, solid carrier, b) contacting said bound HCG receptor protein with a sample solution, c) allowing the immunological reaction between the bound HCG receptor protein and any HCG in the sample solution to take place.
d) washing the solid carrier, e) contacting the bound HCG receptor protein with a known amount of a reagent, said reagent consisting essentially of gold sol particles, said particles having HCG conjugate coupled to their surfaces, to bind said reagent to that part of said bound receptor protein which has undergone the reaction in step (c), f) and determining the amount of gold in the free reagent or the bound reagent which is a measure of the amount of HCG in the solution.
d) washing the solid carrier, e) contacting the bound HCG receptor protein with a known amount of a reagent, said reagent consisting essentially of gold sol particles, said particles having HCG conjugate coupled to their surfaces, to bind said reagent to that part of said bound receptor protein which has undergone the reaction in step (c), f) and determining the amount of gold in the free reagent or the bound reagent which is a measure of the amount of HCG in the solution.
11. Process for the detection and/or determination of one or more components of the reaction between a specific binding protein and the corresponding bindable substance in an aqueous test sample, whilst applying the known binding af-finity of such components for one another, which comprises providing one or more labelled components obtained by coupling directly or indirectly the desired component of said reaction to particles of an aqueous dispersion of a metal, metal com-pound or polymer nuclei coated with a metal or metal compound, having a particle size of at least 5 nm, contacting said aqueous test sample with said one or more labelled components, whereby during the reaction or after an adequate reaction time, the physical properties and/or the amount of the metal and/or the formed metal containing agglomerate is/are deter-mined in the test sample or in a derived fraction thereof, which determination provides a qualitative and/or quantitative indication of the component or components to be detected and/or determined.
12. Process according to claim 11, whereby the component or components of the reaction between a specific binding protein and the corresponding bindable substance are immuno-chemical components, which comprises providing one or more labelled components obtained by coupling directly or indirectly the desired immuno chemical component to particles or an aqueous dispersion of a metal, metal compound or polymer nuclei coated with a metal or metal compound, having a particle size of at least 5 nm, contacting said aqueous test sample with said one or more labelled components, whereby after an adequate reaction time, the physical properties and/or the amount of the metal and/or the formed metal contain-ing agglomerate is determined in the test sample or in a derived fraction thereof, which determina-tion provides a qualitative and/or quantitative indication of the immunological component or components to be detected and/or determined.
13. Process according to claims 11 or 12, wherein said detection and/or determination is carried out after separation of the bound and free labelled components.
14. Process according to claims 11 and 17, characterized in that one or more labelled components are used, obtained by coupling directly or indirectly the desired reactant to par-ticles of a sol, having a particle size varying from 10 to 100 nm.
15. Process according to claims 11 and 12, characterized in that the labelled component is obtained by adding to a sol of a metal, a metal compound or polymer nuclei coated with a metal or metal compound, a certain amount of the immunochemical component to be labelled, which latter completely or partially coats the sol particles, after which further coating may be carried out with an immunochemically inert polar macromolecule.
16. Process according to claims 11 and 12, characterized in that the labelled component is obtained by adding to a sol of a metal, a metal compound or polymer nuclei coated with a metal or a metal compound, one or more immunochemically inert hydrophilic macromolecules, which coat the sol particles, after which the immunochemical component is coupled to the coating material.
17. Process according to claim 11, characterized in that the labelled component is obtained by introducing a metal sol into an environment of monomers, causing these latter to polymerize, or co-polymerize respectively, in situ, so that coating of the sol particles results, and subsequently coupling the immunochemical component to the polymeric material.
18. Process according to claim 17, characterized in that the sol particles are first protected by a hydrophilic macro-molecule, after which (co)-polymerization occurs under the influence of an inorganic initiator.
19. Process according to claims 11-12, characterized by the use of particles of a sol of gold, silver or platinum or compounds of these metals or iron or copper compounds.
20. Process according to claims 11-12, characterized in that, after separation of the bound and the free labelled components, the physical properties and/or the amount of the metal is determined in the fraction containing the bound labelled component.
21. Process according to claims 11-12, characterized in that the immunochemical component or components to be deter-mined is/are allowed to react with one or more insolubilized immunochemical component, before the labelled component or components is/are added.
22. Process according to claims 11-12, characterized in that the bound labelled component or components is/are dis-engaged after separation of the free labelled component or components and the amount of the metal or metals is/are de-termined in the obtained liquid fraction.
23. Process according to claims 11-12, characterized in that two or more immunochemical components are determined in one test sample by use of two or more different labelled com-ponents.
24. Process according to claims 11-12, characterized in that the occurrance of a change in colour or colour intensity of the reaction mixture is observed or measured, which change is a qualitative or quantitative indication of the immuno-chemical component to be determined.
25. Test kit, to be used for the determination of one or more components of the reaction between a specific binding protein and the corresponding bindable substance in an aqueous medium, containing:
a) a metal-labelled component which has been obtained by coupling a component of said reaction to particles of an aqueous dispersion of a metal, metal compound or polymer nuclei coated with a metal or metal compound, which particles have a size of at least 5 nm, b) one or more additional immunochemical reagents of which at least one reagent is a ligand or an immobilized ligand, which ligand is selected from the group consisting of (aa) a ligand capable of binding with the metal-labelled component (a);
(bb) a ligand capable of binding with a binding partner of the metal-labelled component (a);
(cc) a ligand capable of binding with at least one of the component(s) to be determined; and (dd) a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined .
a) a metal-labelled component which has been obtained by coupling a component of said reaction to particles of an aqueous dispersion of a metal, metal compound or polymer nuclei coated with a metal or metal compound, which particles have a size of at least 5 nm, b) one or more additional immunochemical reagents of which at least one reagent is a ligand or an immobilized ligand, which ligand is selected from the group consisting of (aa) a ligand capable of binding with the metal-labelled component (a);
(bb) a ligand capable of binding with a binding partner of the metal-labelled component (a);
(cc) a ligand capable of binding with at least one of the component(s) to be determined; and (dd) a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined .
26. Test kit according to claim 25, of which the aqueous dispersion is a sol, having particles with a size varying from 10-100 nm, which are either directly coated by the component of said reaction or by an inert polymer, to which the desired component has been coupled.
27. Test kit according to claims 25 and 26, of which the labelled component is an immuno component.
28. Test kit according to claim 25, of which the labelled component is freeze-dried.
29. Test kit according to claim 25, of which the freeze-dried reagent contains also a buffer substance.
30. The process for the detection and/or determination of one or more components of the reaction between a specific binding protein and the corresponding bindable substance in an aqueous test sample, by applying the known binding affinity of such components for one another, comprising:
(a) employing one or more labelled components, obtained by coupling directly or indirectly the desired component of said reaction to particles of an aqueous sol dispersion of a metal, metal compound or polymer nuclei coated with a metal or metal compound, having a particle size of at least 5 nm, and (b) detecting and/or determining during the reaction or after an adequate reaction time and optionally after separation of the bound and free labelled components, the physical properties and/or the amount of the metal and/or a formed agglomerate containing said sol dispersed particles in the test sample or one of the derived fractions, which detection and/or determination provides a qualitative and/or quantitative indication of the component or components to be detected and/or determined.
(a) employing one or more labelled components, obtained by coupling directly or indirectly the desired component of said reaction to particles of an aqueous sol dispersion of a metal, metal compound or polymer nuclei coated with a metal or metal compound, having a particle size of at least 5 nm, and (b) detecting and/or determining during the reaction or after an adequate reaction time and optionally after separation of the bound and free labelled components, the physical properties and/or the amount of the metal and/or a formed agglomerate containing said sol dispersed particles in the test sample or one of the derived fractions, which detection and/or determination provides a qualitative and/or quantitative indication of the component or components to be detected and/or determined.
31. The process according to claim 30, whereby the component or components of the reaction between a specific binding protein and the corresponding bindable substance are immunochemical components, such as haptens, antigens, or antibodies, comprising:
(a) employing labelled components, obtained by coupling directly or indirectly the desired immunochemical component to particles of an aqueous sol dispersion of a metal, metal compound or polymer nuclei coated with a metal or metal compound, having a particle size of at least 5 nm, and (b) detecting or determining after an adequate reaction time and optionally after separation of the bound and free labelled components, the physical properties and/or the amount of the metal and/or a formed agglomerate containing said metal particles in the test sample or one of the derived fractions, which detection and/or determination provides a qualitative and/or quantitative indication of the immunological component or components to be detected and/or determined.
(a) employing labelled components, obtained by coupling directly or indirectly the desired immunochemical component to particles of an aqueous sol dispersion of a metal, metal compound or polymer nuclei coated with a metal or metal compound, having a particle size of at least 5 nm, and (b) detecting or determining after an adequate reaction time and optionally after separation of the bound and free labelled components, the physical properties and/or the amount of the metal and/or a formed agglomerate containing said metal particles in the test sample or one of the derived fractions, which detection and/or determination provides a qualitative and/or quantitative indication of the immunological component or components to be detected and/or determined.
32. The process according to claim 30, wherein one or more labelled components are used, obtained by coupling directly or indirectly the desired reactant to particles of a sol, having a particle size varying from 10 to 100 nm.
33. The process according to claim 30 wherein the labelled component is obtained by adding to a sol of a metal, a metal compound or polymer nuclei coated with a metal or metal compound, a certain amount of the immunochemical component to be labelled, which latter component completely or partially coats the sol particles, after which further coating may be carried out with an immunochemically inert polar macromolecule.
34. The process according to claim 30 wherein the labelled component is obtained by adding to a sol of a metal, a metal compound or polymer nuclei coated with a metal or a metal compound, one or more immunochemically inert hydro-philic macromolecules which coat the sol particles, after which the immunochemical component is coupled to the coating material.
35. The process according to claim 30 wherein the labelled component is obtained by:
(a) introducing a metal sol into an environment of monomers, causing said monomers to polymerize or co-polymerize in situ, so that coating of the sol particles results, and (b) subsequently coupling the immunochemical component to the polymeric material.
(a) introducing a metal sol into an environment of monomers, causing said monomers to polymerize or co-polymerize in situ, so that coating of the sol particles results, and (b) subsequently coupling the immunochemical component to the polymeric material.
36. The process according to claim 30 wherein the sol particles are first protected by an inert hydrophilic macro-molecule, after which (CO)-polymerization occurs under the influence of an inorganic initiator.
37. The process according to claim 30 wherein the dis-persed particles are of a sol of gold, silver or platinum, or compounds of these metals, or iron or copper compounds.
38. The process according to claim 30 wherein after separation of the bound and the free labelled components, the physical properties and/or the amount of the metal is determined in the fraction containing the bound labelled component.
39. The process according to claim 30 wherein the immunochemical component or components to be determined is/are allowed to react with one or more insolubilized immunochemical component(s), before the labelled component or components is/
are added.
are added.
40. The process according to claim 30 wherein the bound labelled component or components is/are disengaged after separation of the free labelled component or components and the amount of the metal or metals is/are determined in the obtained liquid fraction.
41. The process according to claim 30 wherein two or more immunochemical components are determined in one test sample by use of two or more different labelled components.
42. The process according to claim 30 wherein the occurrance of a change in color or color intensity of the reaction mixture is observed or measured, which change is a qualitative or quantitative indication of the immunochemical, component to be determined.
43. A test kit, to be used for the detection and/or determination of one or more components of the reaction between a specific binding protein and a corresponding bindable sub-stance thereto in an aqueous medium according to a predetermined protocol, comprising:
(a) a metal-labelled component which has been obtained by coupling a component of said reaction to particles of an aqueous sol dispersion of a metal, metal compound or polymer nuclei coated with a metal or metal compound, which particles have a size of at least 5 nm;
(b) one or more additional immunochemical reagents of which at least one reagent is a ligand or an immobilized ligand, which ligand is selected from the group consisting of (aa) a ligand capable of binding with the metal-labelled component (a);
(bb) a ligand capable of binding with a binding partner of the metal-labelled component (a);
(cc) a ligand capable of binding with at least one of the component(s) to be determined, and (dd) a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined, and (c) directions for the performance of a protocol for the detection and/or determination of one or more components of an immunochemical reaction in an aqueous medium between a specific binding protein and a corresponding bindable substance thereto.
(a) a metal-labelled component which has been obtained by coupling a component of said reaction to particles of an aqueous sol dispersion of a metal, metal compound or polymer nuclei coated with a metal or metal compound, which particles have a size of at least 5 nm;
(b) one or more additional immunochemical reagents of which at least one reagent is a ligand or an immobilized ligand, which ligand is selected from the group consisting of (aa) a ligand capable of binding with the metal-labelled component (a);
(bb) a ligand capable of binding with a binding partner of the metal-labelled component (a);
(cc) a ligand capable of binding with at least one of the component(s) to be determined, and (dd) a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined, and (c) directions for the performance of a protocol for the detection and/or determination of one or more components of an immunochemical reaction in an aqueous medium between a specific binding protein and a corresponding bindable substance thereto.
44. A test kit according to claim 43, in which the aqueous dispersion is a sol, having particles with a size varying from 10-100 nm, which are either directly coated by the component of said reaction or by an inert polymer, to which the desired component has been coupled.
45. The test kit according to claim 43, of which the labelled component is an immuno component.
46. The test kit according to claim 43, of which the labelled component is freeze-dried.
47. The test kit according to claim 43, of which the freeze-dried reagent contains also a buffer substance.
48. A method for the determination of a first component of an immunochemical reaction in an aqueous medium selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm, which particles have attached to their surfaces an immunochemical component which is immuno-chemically equivalent to said first component to be determined, and (2) a known amount of an insolubilized second component capable of reacting with either said first component to be determined or said reagent;
(b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component, and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the metal of the metal sol dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.
(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm, which particles have attached to their surfaces an immunochemical component which is immuno-chemically equivalent to said first component to be determined, and (2) a known amount of an insolubilized second component capable of reacting with either said first component to be determined or said reagent;
(b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component, and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the metal of the metal sol dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.
49. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm, which particles have attached to their surfaces (i) an immunochemical component immuno-chemically equivalent to said first component to be determined, and (ii) an immunochemically inert macromolecule, and (2) a known amount of an insolubilized second immunochemical component capable of reacting with either said first component to be determined or said reagent, (b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced: and (c) determining the amount of the metal of the metal sol dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.
(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm, which particles have attached to their surfaces (i) an immunochemical component immuno-chemically equivalent to said first component to be determined, and (ii) an immunochemically inert macromolecule, and (2) a known amount of an insolubilized second immunochemical component capable of reacting with either said first component to be determined or said reagent, (b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced: and (c) determining the amount of the metal of the metal sol dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.
50. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm, which particles have a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it an immunochemical com-ponent which is immunochemically equivalent to said first com-ponent, and (2) a known amount of an insolubilized second immunochemical component capable of reacting with either said first component to be determined or said reagent;
(b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the metal of the metal sol dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.
(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm, which particles have a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it an immunochemical com-ponent which is immunochemically equivalent to said first com-ponent, and (2) a known amount of an insolubilized second immunochemical component capable of reacting with either said first component to be determined or said reagent;
(b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the metal of the metal sol dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.
51. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first component to be determined with (1) a known amount of reagent consisting essentially of metal sol dispersion having a particle size of at least 5 nm, which sol particles have attached to their surfaces a second immunochemical component of said reaction capable of reacting with said first component to be determined, and (2) a known amount of an insolubilized second immunochemical component which is immunochemically equivalent to said first component and capable of reacting with said reagent, (b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the metal of the metal sol dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.
(a) contacting a sample containing the first component to be determined with (1) a known amount of reagent consisting essentially of metal sol dispersion having a particle size of at least 5 nm, which sol particles have attached to their surfaces a second immunochemical component of said reaction capable of reacting with said first component to be determined, and (2) a known amount of an insolubilized second immunochemical component which is immunochemically equivalent to said first component and capable of reacting with said reagent, (b) allowing sufficient time for reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the metal of the metal sol dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.
52. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent, said reagent consisting essentially of metal sol dispersion particles having sizes of at least 5 nm, which sol particles have attached to their surfaces (i) a second immunochemical component capable of reacting with said first component to be determined, and (ii) an immunochemically inert macromolecule and (2) a known amount of an insolubilized second immunochemical component which is immunochemically equivalent to said first component to be determined and capable of reacting with said reagent;
(b) allowing sufficient time for the reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the metal of the metal sol dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.
(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent, said reagent consisting essentially of metal sol dispersion particles having sizes of at least 5 nm, which sol particles have attached to their surfaces (i) a second immunochemical component capable of reacting with said first component to be determined, and (ii) an immunochemically inert macromolecule and (2) a known amount of an insolubilized second immunochemical component which is immunochemically equivalent to said first component to be determined and capable of reacting with said reagent;
(b) allowing sufficient time for the reaction to take place, whereby (1) a fraction of said first component to be determined bound to said insolubilized component and (2) a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the metal of the metal sol dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.
53. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corres-ponding bindable substance, comprising:
(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm, which particles have a coating of an inert hydrophilic polymer or copolymer, the surface of each coated sol particle having attached to it a second immunochemical component of said reaction capable of reacting with said first component to be determined, and (2) a known amount of an insolubilized third immunochemical component which is immuno-chemically equivalent to said first component and capable of reacting with said reagent;
(b) allowing sufficient time for the reaction to take place, whereby a fraction of said first component to be determined bound to said insolubilized component and a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the metal of the metal sol dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.
(a) contacting a sample containing the first component to be determined with (1) a known amount of a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm, which particles have a coating of an inert hydrophilic polymer or copolymer, the surface of each coated sol particle having attached to it a second immunochemical component of said reaction capable of reacting with said first component to be determined, and (2) a known amount of an insolubilized third immunochemical component which is immuno-chemically equivalent to said first component and capable of reacting with said reagent;
(b) allowing sufficient time for the reaction to take place, whereby a fraction of said first component to be determined bound to said insolubilized component and a fraction of said first component to be determined free from said insolubilized component are produced; and (c) determining the amount of the metal of the metal sol dispersion particles in one of said fractions, which is a measure of the amount of said first component to be determined in said sample.
54. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of its corresponding bindable substance, comprising:
(a) binding an amount of a second immunochemical component of said reaction capable of reacting with said first component to be determined, to the surface of a water-insoluble water-insuspensible, solid carrier, (b) contacting said bound second component with a sample containing the first component to be determined;
(c) allowing sufficient time for reaction to take place;
(d) contacting said bound reaction product of (c) with a known amount of a reagent consisting essentially of metal dispersion sol particles having a particle size of at least 5 nm, which sol particles have attached to their surface an immuno-chemical component capable of reacting with said first component to be determined;
(e) allowing sufficient time for reaction to take place, to bind a fraction of said reagent to that part of said bound second immunochemical component which has undergone the reaction in step (c), leaving a remaining fraction of said reagent free and not bound; and (f) determining the amount of the metal sol dispersion particles in the free reagent fraction or the bound reagent fraction, which is a measure of the amount of said first component to be determined in said sample.
(a) binding an amount of a second immunochemical component of said reaction capable of reacting with said first component to be determined, to the surface of a water-insoluble water-insuspensible, solid carrier, (b) contacting said bound second component with a sample containing the first component to be determined;
(c) allowing sufficient time for reaction to take place;
(d) contacting said bound reaction product of (c) with a known amount of a reagent consisting essentially of metal dispersion sol particles having a particle size of at least 5 nm, which sol particles have attached to their surface an immuno-chemical component capable of reacting with said first component to be determined;
(e) allowing sufficient time for reaction to take place, to bind a fraction of said reagent to that part of said bound second immunochemical component which has undergone the reaction in step (c), leaving a remaining fraction of said reagent free and not bound; and (f) determining the amount of the metal sol dispersion particles in the free reagent fraction or the bound reagent fraction, which is a measure of the amount of said first component to be determined in said sample.
55. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) binding an amount of a second immunochemical component capable of reacting with said first component to be determined, to the surface of a water-insoluble, water-insuspensible, solid carrier, (b) contacting said bound second component with a sample containing the first component to be determined, (c) allowing sufficient time for reaction to take place;
(d) contacting said bound reaction product of (c) with a known amount of reagent consisting essentially of metal sol dispersion particles, having a particle size of at least 5 nm, said sol particles having attached to their surfaces (i) an immunochemical component capable of reacting with said first component to be determined, and (ii) an immunochemically inert macromolecule;
(e) allowing sufficient time for reaction to take place to bind a fraction of said reagent to that part of said bound second component which has undergone the reaction in step (c), leaving a remaining fraction of said reagent free and not bound: and (f) determining the amount of the metal sol dispersion particles in the free reagent fraction or the bound reagent fraction, which is a measure of the amount of said component to be determined in said sample.
(a) binding an amount of a second immunochemical component capable of reacting with said first component to be determined, to the surface of a water-insoluble, water-insuspensible, solid carrier, (b) contacting said bound second component with a sample containing the first component to be determined, (c) allowing sufficient time for reaction to take place;
(d) contacting said bound reaction product of (c) with a known amount of reagent consisting essentially of metal sol dispersion particles, having a particle size of at least 5 nm, said sol particles having attached to their surfaces (i) an immunochemical component capable of reacting with said first component to be determined, and (ii) an immunochemically inert macromolecule;
(e) allowing sufficient time for reaction to take place to bind a fraction of said reagent to that part of said bound second component which has undergone the reaction in step (c), leaving a remaining fraction of said reagent free and not bound: and (f) determining the amount of the metal sol dispersion particles in the free reagent fraction or the bound reagent fraction, which is a measure of the amount of said component to be determined in said sample.
56. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) binding an amount of a second immunochemical component capable of reacting with said first component to be determined, to the surface of a water-insoluble, water-insuspensible, solid carrier:
(b) contacting said bound second component with a sample containing the first component to be determined;
(c) allowing sufficient time for reaction to take place;
(d) contacting said bound reaction product of (c) with a known amount of reagent consisting essentially of metal sol dispersion particles, having a particle size of at least 5 nm and having a coating of an inert hydrophilic polymer or co-polymer, the surface of each coated particle having attached to it an immunochemical component capable of reacting with said first component to be determined;
(e) allowing sufficient time for reaction to take place to bind a fraction of said reagent to that part of said bound second component which has undergone the reaction in step (c), leaving a remaining fraction of said reagent free and not bound; and (f) determining the amount of the metal sol dispersion particles in the free reagent fraction or the bound reagent fraction, which is a measure of the amount of said first component to be determined in said sample.
(a) binding an amount of a second immunochemical component capable of reacting with said first component to be determined, to the surface of a water-insoluble, water-insuspensible, solid carrier:
(b) contacting said bound second component with a sample containing the first component to be determined;
(c) allowing sufficient time for reaction to take place;
(d) contacting said bound reaction product of (c) with a known amount of reagent consisting essentially of metal sol dispersion particles, having a particle size of at least 5 nm and having a coating of an inert hydrophilic polymer or co-polymer, the surface of each coated particle having attached to it an immunochemical component capable of reacting with said first component to be determined;
(e) allowing sufficient time for reaction to take place to bind a fraction of said reagent to that part of said bound second component which has undergone the reaction in step (c), leaving a remaining fraction of said reagent free and not bound; and (f) determining the amount of the metal sol dispersion particles in the free reagent fraction or the bound reagent fraction, which is a measure of the amount of said first component to be determined in said sample.
57. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first component to be determined with a known amount of a reagent, said reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm, said sol particles having attached to their surfaces a second immunochemical com-ponent of said reaction capable of reacting with said first component to be determined;
(b) allowing sufficient time for reaction to take place; and (c) determining the color of the reaction solu-tion, which is a measure of the amount of said first component to be determined in said sample.
(a) contacting a sample containing the first component to be determined with a known amount of a reagent, said reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm, said sol particles having attached to their surfaces a second immunochemical com-ponent of said reaction capable of reacting with said first component to be determined;
(b) allowing sufficient time for reaction to take place; and (c) determining the color of the reaction solu-tion, which is a measure of the amount of said first component to be determined in said sample.
58. A method for the determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first component to be determined with a known amount of a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm, said sol particles having attached to their surfaces (i) a second immunochemical component of said reaction capable of reacting with said first component to be determined, and (ii) an immunochemically inert macro-molecule, (b) allowing sufficient time for a reaction to take place, and (c) determining the color of the reaction solution, which is a measure of the amount of said first com-ponent to be determined in said sample.
(a) contacting a sample containing the first component to be determined with a known amount of a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm, said sol particles having attached to their surfaces (i) a second immunochemical component of said reaction capable of reacting with said first component to be determined, and (ii) an immunochemically inert macro-molecule, (b) allowing sufficient time for a reaction to take place, and (c) determining the color of the reaction solution, which is a measure of the amount of said first com-ponent to be determined in said sample.
59. A method for the determination of a first com-ponent of an immunochemical reaction in an aqueous test medium selected from the group consisting of a specific binding protein and its corresponding bindable substance, comprising:
(a) contacting a sample containing the first component to be determined with a known amount of a reagent consisting of metal sol dispersion particles having a particle size of at least 5 nm, said sol particles having a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it a second immunochemical component of said reaction capable of reacting with said first component to be determined;
(b) allowing sufficient time for a reaction to take place; and (c) determining the color of the reaction solution, which is a measure of the amount of said first component to be determined in said sample.
(a) contacting a sample containing the first component to be determined with a known amount of a reagent consisting of metal sol dispersion particles having a particle size of at least 5 nm, said sol particles having a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it a second immunochemical component of said reaction capable of reacting with said first component to be determined;
(b) allowing sufficient time for a reaction to take place; and (c) determining the color of the reaction solution, which is a measure of the amount of said first component to be determined in said sample.
60. A method as in one of claims 48-50, wherein at least one of said components is drawn from the group consist-ing of antigens, haptens, and antibodies.
61. A method as in claim 48, wherein the metal sol dispersion particles are drawn from the group consisting of particles of platinum, gold, silver, copper, silver iodide, silver bromide, copper hydrous oxide, iron hydrous oxide, chromium hydroxide, aluminum hydroxide, aluminum hydrous oxide, chromium hydrous oxide, platinum, silver iodide, iron oxide, aluminum hydroxide, lead sulphide, barium sulfate, titanium dioxide, vanadium oxide, iron hydroxide, arsenic sulphide, manganese hydroxide and mercury sulphide.
62. A method as in claim 61, in which the metal sol dispersion particles are particles of gold.
63. A method for the immunochemical determination of human placental lactogen (HPL), comprising:
(a) binding a known amount of rabbit anti-HPL
immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound immunoglobulin with a sample solution containing the HPL to be determined;
(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and said HPL in the sample solution in step (b) to take place, to bind the HPL to be determined to the insolubilized rabbit anti- HPL, forming a first solid phase, and leaving a first liquid phase, (d) separating the first liquid and solid phases, (e) contacting said first reacted solid phase and bound HPL therein with a known amount of a reagent, said reagent consisting essentially of gold sol dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HPL immunoglobulin attached to their surfaces, (f) allowing sufficient time for a second immunological reaction to take place, to bind said reagent to that part of said HPL solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase, (g) separating the second solid phase from the second liquid phase, and (h) determining the amount of gold sol in the second liquid phase or the second solid phase, which is a measure of the amount of HPL in said sample solution.
(a) binding a known amount of rabbit anti-HPL
immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound immunoglobulin with a sample solution containing the HPL to be determined;
(c) allowing sufficient time for an immunological reaction between the bound immunoglobulin and said HPL in the sample solution in step (b) to take place, to bind the HPL to be determined to the insolubilized rabbit anti- HPL, forming a first solid phase, and leaving a first liquid phase, (d) separating the first liquid and solid phases, (e) contacting said first reacted solid phase and bound HPL therein with a known amount of a reagent, said reagent consisting essentially of gold sol dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HPL immunoglobulin attached to their surfaces, (f) allowing sufficient time for a second immunological reaction to take place, to bind said reagent to that part of said HPL solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase, (g) separating the second solid phase from the second liquid phase, and (h) determining the amount of gold sol in the second liquid phase or the second solid phase, which is a measure of the amount of HPL in said sample solution.
64. A method for the immunochemical determination of hepatitis Surface B antigen (HBsAg), comprising:
(a) binding a known amount of sheep anti-HBsAg immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound immunoglobulin with a sample solution containing the HBsAg to be determined, (c) allowing sufficient time for an immuno-logical reaction between the bound immunoglobulin and the HBsAg in the sample solution in step (b) to take place, to bind the HBsAg to be determined to the insolubilized sheep anti-HBsAg, forming a first solid phase, and leaving a first liquid phase, (d) separating the first liquid and solid phases, (e) contacting said first reacted solid phase and bound HBsAg therein with a known amount of a reagent having a particle size of at least 5 nm, said reagent consisting essentially of gold sol particles, said sol particles having anti-HBsAg immunoglobulin attached to their surfaces;
(f) allowing sufficient time for a second immunochemical reaction to take place, to bind said reagent to that part of said HBsAg solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of gold sol in the second liquid phase or the second solid phase, which is a measure of the amount of HBsAg in said sample solution.
(a) binding a known amount of sheep anti-HBsAg immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound immunoglobulin with a sample solution containing the HBsAg to be determined, (c) allowing sufficient time for an immuno-logical reaction between the bound immunoglobulin and the HBsAg in the sample solution in step (b) to take place, to bind the HBsAg to be determined to the insolubilized sheep anti-HBsAg, forming a first solid phase, and leaving a first liquid phase, (d) separating the first liquid and solid phases, (e) contacting said first reacted solid phase and bound HBsAg therein with a known amount of a reagent having a particle size of at least 5 nm, said reagent consisting essentially of gold sol particles, said sol particles having anti-HBsAg immunoglobulin attached to their surfaces;
(f) allowing sufficient time for a second immunochemical reaction to take place, to bind said reagent to that part of said HBsAg solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of gold sol in the second liquid phase or the second solid phase, which is a measure of the amount of HBsAg in said sample solution.
65. A method for the immunochemical determination of testosterone in a liquid sample, comprising:
(a) binding a known amount of rabbit anti-testosterone immunoglobulin to the surface of a water-insoluble, water-insuspensible, solid carrier;
(b) contacting said bound rabbit anti-testoster-one with a sample solution containing the testosterone to be determined;
(c) allowing sufficient time for an immuno-logical reaction between the bound rabbit anti-testosterone immunoglobulin and the testosterone in the sample solution in step (b) to take place to bind the testosterone to be determined to the insolubilized rabbit anti-testosterone, forming a first solid phase, and leaving a first liquid phase;
(d) separating the first liquid and solid phases;
(e) contacting said first reacted solid phase and bound testosterone therein with a known amount of a reagent having a particle size of at least 5 nm, said reagent consisting essentially of silver sol dispersion particles, said sol particles having testosterone-ll.alpha.-hemisuccinyl-bovine serum albumin attached to their surfaces, to form a silver particle-testosterone-ll.alpha.-hemisuccinyl-bovine serum albumin conjugate, (f) allowing sufficient time for a second immunochemical reaction to take place to bind said reagent to that part of said solid phase bound rabbit anti-testosterone immunoglobulin which has not undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of silver sol in the second solid phase or the second liquid phase, which is a measure of the amount of testosterone in said liquid sample.
(a) binding a known amount of rabbit anti-testosterone immunoglobulin to the surface of a water-insoluble, water-insuspensible, solid carrier;
(b) contacting said bound rabbit anti-testoster-one with a sample solution containing the testosterone to be determined;
(c) allowing sufficient time for an immuno-logical reaction between the bound rabbit anti-testosterone immunoglobulin and the testosterone in the sample solution in step (b) to take place to bind the testosterone to be determined to the insolubilized rabbit anti-testosterone, forming a first solid phase, and leaving a first liquid phase;
(d) separating the first liquid and solid phases;
(e) contacting said first reacted solid phase and bound testosterone therein with a known amount of a reagent having a particle size of at least 5 nm, said reagent consisting essentially of silver sol dispersion particles, said sol particles having testosterone-ll.alpha.-hemisuccinyl-bovine serum albumin attached to their surfaces, to form a silver particle-testosterone-ll.alpha.-hemisuccinyl-bovine serum albumin conjugate, (f) allowing sufficient time for a second immunochemical reaction to take place to bind said reagent to that part of said solid phase bound rabbit anti-testosterone immunoglobulin which has not undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of silver sol in the second solid phase or the second liquid phase, which is a measure of the amount of testosterone in said liquid sample.
66. A method for the immunochemical determination of the titer of human anti-Rubella sera, comprising:
(a) binding a known amount of Rubella antigen to the surface of a water-insoluble, water-insuspensible, solid carrier;
(b) contacting said bound Rubella antigen with a human serum sample containing the titer of human anti-Rubella sera to be determined;
(c) allowing sufficient time for an immuno-logical reaction between the bound Rubella antigen and the serum containing the titer of human anti-Rubella sera to be determined to take place to bind the anti-Rubella sera to the insolubilized Rubella antigen, forming a first solid phase, and leaving a first liquid phase;
(d) separating the first liquid and solid phases;
(e) contacting said first reacted solid phase and bound anti-Rubella titer therein with a known amount of a reagent, said reagent consisting essentially of gold sol dis-persion particles having a particle size of at least 5 nm, said sol particles having sheep anti-human immunoglobulin attached to their surfaces to form a gold particle sheep antihuman immunoglobulin conjugate, (f) allowing sufficient time for a second immunological reaction to take place to bind said reagent to the bound anti-Rubella titer solid phase, resulting in the binding of said reagent to that part of said bound antigen which has undergone the reaction in step (c), to form a second solid and a second liquid phase, (g) separating the second liquid and solid phases; and (h) determining the amount of gold sol in the second solid phase or the second liquid phase, which is a measure of the titer of the human anti-Rubella serum to be determined in said sample.
(a) binding a known amount of Rubella antigen to the surface of a water-insoluble, water-insuspensible, solid carrier;
(b) contacting said bound Rubella antigen with a human serum sample containing the titer of human anti-Rubella sera to be determined;
(c) allowing sufficient time for an immuno-logical reaction between the bound Rubella antigen and the serum containing the titer of human anti-Rubella sera to be determined to take place to bind the anti-Rubella sera to the insolubilized Rubella antigen, forming a first solid phase, and leaving a first liquid phase;
(d) separating the first liquid and solid phases;
(e) contacting said first reacted solid phase and bound anti-Rubella titer therein with a known amount of a reagent, said reagent consisting essentially of gold sol dis-persion particles having a particle size of at least 5 nm, said sol particles having sheep anti-human immunoglobulin attached to their surfaces to form a gold particle sheep antihuman immunoglobulin conjugate, (f) allowing sufficient time for a second immunological reaction to take place to bind said reagent to the bound anti-Rubella titer solid phase, resulting in the binding of said reagent to that part of said bound antigen which has undergone the reaction in step (c), to form a second solid and a second liquid phase, (g) separating the second liquid and solid phases; and (h) determining the amount of gold sol in the second solid phase or the second liquid phase, which is a measure of the titer of the human anti-Rubella serum to be determined in said sample.
67. A method for the immunochemical determination of HCG, comprising:
(a) binding a known amount of a rabbit anti-HCG
immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound rabbit anti-HCG-immunoglobulin with a sample solution containing the HCG to be determined:
(c) allowing sufficient time for an immuno-logical reaction between the rabbit anti-HCG immunoglobulin and HCG in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG immunoglobulin, forming a first solid phase, and leaving a first liquid phase;
(d) separating the first solid phase from the first liquid phase;
(e) contacting said first solid phase and bound HCG with a known amount of a reagent consisting essentially of iron oxide sol dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immuno-globulin attached to their surfaces;
(f) allowing sufficient time for a second immunological reaction to occur to bind said reagent to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid and second liquid phases; and (h) determining the amount of iron sol in the second solid phase or the second liquid phase, which is a measure of the amount of the HCG to be determined in said sample solution.
(a) binding a known amount of a rabbit anti-HCG
immunoglobulin to the surface of a water-insoluble, water-insuspensible solid carrier;
(b) contacting said bound rabbit anti-HCG-immunoglobulin with a sample solution containing the HCG to be determined:
(c) allowing sufficient time for an immuno-logical reaction between the rabbit anti-HCG immunoglobulin and HCG in the sample solution in step (b) to take place, to bind the HCG to be determined to the insolubilized rabbit anti-HCG immunoglobulin, forming a first solid phase, and leaving a first liquid phase;
(d) separating the first solid phase from the first liquid phase;
(e) contacting said first solid phase and bound HCG with a known amount of a reagent consisting essentially of iron oxide sol dispersion particles having a particle size of at least 5 nm, said particles having rabbit anti-HCG immuno-globulin attached to their surfaces;
(f) allowing sufficient time for a second immunological reaction to occur to bind said reagent to that part of said HCG solid phase bound immunoglobulin which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid and second liquid phases; and (h) determining the amount of iron sol in the second solid phase or the second liquid phase, which is a measure of the amount of the HCG to be determined in said sample solution.
68. A method for the immunochemical determination of HCG, comprising:
(a) contacting an aqueous sample containing the HCG to be determined with a known amount of a reagent consisting essentially of gold sol dispersion particles having a particle size of at least 5 nm, said sol particles having rabbit anti-HCG immunoglobulin attached to their surfaces;
(b) allowing sufficient time for an immuno-logical reaction between said reagent and any HCG in the sample solution to take place; and (c) determining the color of the sample solution, which is a measure of the amount of HCG to be determined in said sample solution.
(a) contacting an aqueous sample containing the HCG to be determined with a known amount of a reagent consisting essentially of gold sol dispersion particles having a particle size of at least 5 nm, said sol particles having rabbit anti-HCG immunoglobulin attached to their surfaces;
(b) allowing sufficient time for an immuno-logical reaction between said reagent and any HCG in the sample solution to take place; and (c) determining the color of the sample solution, which is a measure of the amount of HCG to be determined in said sample solution.
69. A method for the determination of HCG, comprising:
(a) binding a known amount of HCG receptor protein to the surface of a water-insoluble, water-insuspensible solid carrier:
(b) contacting said bound receptor protein with a sample solution containing the HCG to be determined:
(c) allowing sufficient time for an immuno-logical reaction between the insolubilized HCG receptor protein and the HCG to be determined:
(d) adding a known amount of reagent to said sample solution, said reagent consisting essentially of gold sol dispersion particles having a particle size of at least 5 nm and having HCG attached to their surfaces:
(e) allowing sufficient time for a reaction to take place between (1) the gold sol dispersion reagent and (2) the insolubilized HCG receptor protein not bound to HCG, to form a solid phase containing a fraction of gold sol dispersion reagent bound to insolubilized HCG receptor protein, and a liquid phase of unbound and free gold dispersion reagent:
(f) separating the liquid and solid phases, and (g) determining the amount of gold sol in either the solid or liquid phase, which is a measure of the amount of HCG in said sample solution.
(a) binding a known amount of HCG receptor protein to the surface of a water-insoluble, water-insuspensible solid carrier:
(b) contacting said bound receptor protein with a sample solution containing the HCG to be determined:
(c) allowing sufficient time for an immuno-logical reaction between the insolubilized HCG receptor protein and the HCG to be determined:
(d) adding a known amount of reagent to said sample solution, said reagent consisting essentially of gold sol dispersion particles having a particle size of at least 5 nm and having HCG attached to their surfaces:
(e) allowing sufficient time for a reaction to take place between (1) the gold sol dispersion reagent and (2) the insolubilized HCG receptor protein not bound to HCG, to form a solid phase containing a fraction of gold sol dispersion reagent bound to insolubilized HCG receptor protein, and a liquid phase of unbound and free gold dispersion reagent:
(f) separating the liquid and solid phases, and (g) determining the amount of gold sol in either the solid or liquid phase, which is a measure of the amount of HCG in said sample solution.
70. A method for the immunochemical determination of HCG, comprising:
(a) binding a known amount of HCG receptor protein to the surface of a water-insoluble, water-insuspensible solid carrier:
(b) contacting said bound receptor protein with a sample solution containing the unknown HCG to be determined:
(c) allowing sufficient time for an immuno-chemical reaction between the bound receptor protein and the HCG in the sample solution to take place, to bind the HCG
to be determined to the insolubilized HCG receptor protein, forming a first solid phase, and leaving a first liquid phase;
(d) separating the first solid phase from the first liquid phase, (e) contacting said first solid phase and insolubilized HCG therein with a known amount of a reagent consisting essentially of gold sol dispersion particles having rabbit anti-HCG immunoglobulin attached to their surfaces having a particle size of at least 5 nm;
(f) allowing sufficient time for a second immunological reaction to occur to bind said reagent to that part of said insolubilized HCG which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of gold sol in the second solid phase or the second liquid phase, which is a measure of the HCG to be determined in said sample solution.
(a) binding a known amount of HCG receptor protein to the surface of a water-insoluble, water-insuspensible solid carrier:
(b) contacting said bound receptor protein with a sample solution containing the unknown HCG to be determined:
(c) allowing sufficient time for an immuno-chemical reaction between the bound receptor protein and the HCG in the sample solution to take place, to bind the HCG
to be determined to the insolubilized HCG receptor protein, forming a first solid phase, and leaving a first liquid phase;
(d) separating the first solid phase from the first liquid phase, (e) contacting said first solid phase and insolubilized HCG therein with a known amount of a reagent consisting essentially of gold sol dispersion particles having rabbit anti-HCG immunoglobulin attached to their surfaces having a particle size of at least 5 nm;
(f) allowing sufficient time for a second immunological reaction to occur to bind said reagent to that part of said insolubilized HCG which has undergone the reaction in step (c), to form a second solid phase and a second liquid phase;
(g) separating the second solid phase from the second liquid phase; and (h) determining the amount of gold sol in the second solid phase or the second liquid phase, which is a measure of the HCG to be determined in said sample solution.
71. Test kit, to be used for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consist-ing of a specific binding protein and a corresponding bindable substance thereto, according to a predetermined protocol, comprising:
(a) a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm and having attached to their surfaces either a component immunochemically similar to said first component to be determined, or a binding partner of the first component, and (b) directions for the performance of a protocol for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and a corresponding bindable substance thereto, using said reagent (a).
(a) a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm and having attached to their surfaces either a component immunochemically similar to said first component to be determined, or a binding partner of the first component, and (b) directions for the performance of a protocol for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and a corresponding bindable substance thereto, using said reagent (a).
72. Test kit, to be used for the detection and/or determination of a first component of an immunochemical eaction in an aqueous medium, selected from the group con-sisting of a specific binding protein and a corresponding bind-able substance thereto, according to a predetermined protocol, comprising:
(a) a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm and having attached to their surfaces (1) either a component immunochemically similar to said first component, or a binding partner to said first component, and (2) an immunochemically inert macromolecule, and (b) directions for the performance of a protocol for the detection and/or determination of first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and a correspond-ing bindable substance thereto, using said reagent (a).
(a) a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm and having attached to their surfaces (1) either a component immunochemically similar to said first component, or a binding partner to said first component, and (2) an immunochemically inert macromolecule, and (b) directions for the performance of a protocol for the detection and/or determination of first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and a correspond-ing bindable substance thereto, using said reagent (a).
73. Test kit, to be used for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consist-ing of a specific binding protein and a corresponding bindable substance thereto, according to a predetermined protocol, comprising:
(a) a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm and having attached to their surfaces a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it a component selected from the group consisting of a component immunochemically equivalent to said first component, or a binding partner to said first component, and (b) directions for the performance of a protocol for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and a corresponding bindable substance thereto, using said reagent (a).
(a) a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm and having attached to their surfaces a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it a component selected from the group consisting of a component immunochemically equivalent to said first component, or a binding partner to said first component, and (b) directions for the performance of a protocol for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and a corresponding bindable substance thereto, using said reagent (a).
74. Test kit according to claim 71, 72 or 73, in which the dispersion is a gold, silver or iron oxide sol.
75. Test kit, according to claim 71, in which the particles range from 10 - 100 nm in size.
76. Test kit according to claim 71 also containing an insolubilized immunocomponent, in which the immunocomponent is selected from the group consisting of (1) a component immunochemically similar to the component to be determined, or (2) a binding partner of the component to be determined.
77. Test kit according to claim 76, in which said reagent containing metal dispersed particles is freeze-dried.
78. Test kit according to claim 77, in which a buffer is included with said reagent.
79- A method as in claims 48, 49 or 50, in which said particles are gold, silver, or iron oxide sols.
80. A method as in claim 48, in which said particles range in size from 10 - 100 nm.
81. Test kit, to be used for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consist-ing of a specific binding protein and a corresponding bindable substance thereto, according to a predetermined protocol, comprising:
(a) a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm and having attached to their surfaces either a component immunochemically similar to said first component to be determined, or a binding partner of the first component, and (b) directions for the performance of a protocol for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and a corresponding bindable substance thereto, using said reagent (a), which protocol comprises:
(1) contacting an aqueous sample having a first color and containing the component to be detected and/or determined with a solution containing reagent (a) for a sufficient time to allow an immunochemical reaction between said component to be detected and/or determined and said reagent (a) to take place, to form a solution of formed agglomerate containing said dispersed particles whereby the reacted solution has a second color, and (2) detecting and/or determining the presence or amount of said agglomerate by observing the difference between the first color and the second color.
(a) a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm and having attached to their surfaces either a component immunochemically similar to said first component to be determined, or a binding partner of the first component, and (b) directions for the performance of a protocol for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and a corresponding bindable substance thereto, using said reagent (a), which protocol comprises:
(1) contacting an aqueous sample having a first color and containing the component to be detected and/or determined with a solution containing reagent (a) for a sufficient time to allow an immunochemical reaction between said component to be detected and/or determined and said reagent (a) to take place, to form a solution of formed agglomerate containing said dispersed particles whereby the reacted solution has a second color, and (2) detecting and/or determining the presence or amount of said agglomerate by observing the difference between the first color and the second color.
82. Test kit, to be used for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and a corresponding bindable substance thereto, according to a predetermined protocol, comprising:
(a) a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm and having attached to their surfaces (1) either a component immunochemically similar to said first component, or a binding partner to said first component, and (2) an immunochemically inert macromolecule, and (b) directions for the performance of a protocol for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and a corresponding bindable substance thereto, using said reagent (a), which protocol comprises:
(1) contacting an aqueous sample having a first color containing the component to be detected and/or determined with a solution containing reagent (a) for a sufficient time to allow an immunochemical reaction between said component to be detected and/or determined and said reagent (a) to take place to form a solution of formed agglomerate containing said dispersed particles whereby the reacted solution has a second color, and (2) detecting and/or determining the presence or amount of said agglomerate by observing the difference between the first color and the second color.
(a) a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm and having attached to their surfaces (1) either a component immunochemically similar to said first component, or a binding partner to said first component, and (2) an immunochemically inert macromolecule, and (b) directions for the performance of a protocol for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and a corresponding bindable substance thereto, using said reagent (a), which protocol comprises:
(1) contacting an aqueous sample having a first color containing the component to be detected and/or determined with a solution containing reagent (a) for a sufficient time to allow an immunochemical reaction between said component to be detected and/or determined and said reagent (a) to take place to form a solution of formed agglomerate containing said dispersed particles whereby the reacted solution has a second color, and (2) detecting and/or determining the presence or amount of said agglomerate by observing the difference between the first color and the second color.
83. Test kit, to be used for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and a corresponding bindable substance thereto, according to a predetermined protocol, comprising:
(a) a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm and having attached to their surfaces a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it a component selected from the group consisting of a component immunochemically equivalent to said first component, or a binding partner to said first com-ponent; and (b) directions for the performance of a protocol for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and a corresponding bindable substance thereto, using said reagent (a), which protocol comprises:
(1) contacting an aqueous sample having a first color containing the component to be detected and/or determined with a solution containing reagent (a) for a sufficient time to allow an immunochemical reaction between said component to be detected and/or determined and said reagent (a) to take place, to form a solution of formed agglomerate containing said dispersed particles whereby the reacted solution has a second color; and (2) detecting and/or determining the presence or amount of said agglomerate by observing the difference between the first color and the second color.
(a) a reagent consisting essentially of metal sol dispersion particles having a particle size of at least 5 nm and having attached to their surfaces a coating of an inert hydrophilic polymer or copolymer, the surface of each coated particle having attached to it a component selected from the group consisting of a component immunochemically equivalent to said first component, or a binding partner to said first com-ponent; and (b) directions for the performance of a protocol for the detection and/or determination of a first component of an immunochemical reaction in an aqueous medium, selected from the group consisting of a specific binding protein and a corresponding bindable substance thereto, using said reagent (a), which protocol comprises:
(1) contacting an aqueous sample having a first color containing the component to be detected and/or determined with a solution containing reagent (a) for a sufficient time to allow an immunochemical reaction between said component to be detected and/or determined and said reagent (a) to take place, to form a solution of formed agglomerate containing said dispersed particles whereby the reacted solution has a second color; and (2) detecting and/or determining the presence or amount of said agglomerate by observing the difference between the first color and the second color.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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NL78.07532 | 1978-07-13 | ||
NL7807532A NL7807532A (en) | 1978-07-13 | 1978-07-13 | METAL IMMUNO TEST. |
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Publication Number | Publication Date |
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CA1135183A true CA1135183A (en) | 1982-11-09 |
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Application Number | Title | Priority Date | Filing Date |
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CA000331816A Expired CA1135183A (en) | 1978-07-13 | 1979-07-13 | Dispersed metal particle labelled assay |
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US (1) | US4313734A (en) |
EP (1) | EP0007654B1 (en) |
JP (1) | JPS5515100A (en) |
AT (1) | ATE786T1 (en) |
AU (1) | AU530217B2 (en) |
CA (1) | CA1135183A (en) |
DE (1) | DE2962333D1 (en) |
DK (1) | DK159799C (en) |
ES (1) | ES482488A1 (en) |
FI (1) | FI77120C (en) |
GR (1) | GR73524B (en) |
HU (1) | HU181659B (en) |
IE (1) | IE48467B1 (en) |
IL (1) | IL57722A (en) |
MX (1) | MX164526B (en) |
NL (1) | NL7807532A (en) |
PH (1) | PH16420A (en) |
PT (1) | PT69900A (en) |
ZA (1) | ZA793276B (en) |
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JPS5745454A (en) * | 1980-09-02 | 1982-03-15 | Fuji Photo Film Co Ltd | Immunochemical measuring method for various minor components |
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GB2095258B (en) * | 1981-03-19 | 1984-09-05 | Janssen Pharmaceutica Nv | A bright field light microscopic method of localizing tissue antigens |
JPS5832167A (en) * | 1981-08-20 | 1983-02-25 | Sekisui Chem Co Ltd | Immunochemical measuring method |
US4454233A (en) * | 1981-10-21 | 1984-06-12 | Wang Associates | Method of tagged immunoassay |
GB8331514D0 (en) * | 1983-11-25 | 1984-01-04 | Janssen Pharmaceutica Nv | Visualization method |
US4703017C1 (en) * | 1984-02-14 | 2001-12-04 | Becton Dickinson Co | Solid phase assay with visual readout |
US4752567A (en) * | 1984-06-21 | 1988-06-21 | Janssen Pharmaceutica N.V. | Method of visualizing individual submicroscopic metal particles |
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1978
- 1978-07-13 NL NL7807532A patent/NL7807532A/en not_active Application Discontinuation
-
1979
- 1979-06-29 EP EP79200352A patent/EP0007654B1/en not_active Expired
- 1979-06-29 DE DE7979200352T patent/DE2962333D1/en not_active Expired
- 1979-06-29 AT AT79200352T patent/ATE786T1/en active
- 1979-07-02 ZA ZA793276A patent/ZA793276B/en unknown
- 1979-07-04 IL IL57722A patent/IL57722A/en unknown
- 1979-07-10 PT PT69900A patent/PT69900A/en unknown
- 1979-07-10 PH PH22787A patent/PH16420A/en unknown
- 1979-07-11 AU AU48834/79A patent/AU530217B2/en not_active Expired
- 1979-07-11 GR GR59571A patent/GR73524B/el unknown
- 1979-07-11 FI FI792179A patent/FI77120C/en not_active IP Right Cessation
- 1979-07-12 JP JP8867879A patent/JPS5515100A/en active Pending
- 1979-07-12 HU HU79AO482A patent/HU181659B/en unknown
- 1979-07-12 MX MX178465A patent/MX164526B/en unknown
- 1979-07-13 US US06/057,309 patent/US4313734A/en not_active Expired - Lifetime
- 1979-07-13 CA CA000331816A patent/CA1135183A/en not_active Expired
- 1979-07-13 ES ES482488A patent/ES482488A1/en not_active Expired
- 1979-07-13 DK DK295679A patent/DK159799C/en not_active IP Right Cessation
- 1979-08-08 IE IE1315/79A patent/IE48467B1/en not_active IP Right Cessation
Also Published As
Publication number | Publication date |
---|---|
IE48467B1 (en) | 1985-02-06 |
DK159799C (en) | 1991-05-06 |
PT69900A (en) | 1979-08-01 |
MX164526B (en) | 1992-08-24 |
FI792179A (en) | 1980-01-14 |
HU181659B (en) | 1983-10-28 |
DE2962333D1 (en) | 1982-04-29 |
ES482488A1 (en) | 1980-08-16 |
IE791315L (en) | 1980-01-13 |
FI77120B (en) | 1988-09-30 |
GR73524B (en) | 1984-03-09 |
IL57722A0 (en) | 1979-11-30 |
ATE786T1 (en) | 1982-04-15 |
PH16420A (en) | 1983-10-03 |
JPS5515100A (en) | 1980-02-01 |
AU530217B2 (en) | 1983-07-07 |
EP0007654A1 (en) | 1980-02-06 |
DK295679A (en) | 1980-01-14 |
NL7807532A (en) | 1980-01-15 |
ZA793276B (en) | 1980-07-30 |
DK159799B (en) | 1990-12-03 |
AU4883479A (en) | 1980-01-17 |
FI77120C (en) | 1989-01-10 |
EP0007654B1 (en) | 1982-03-24 |
US4313734A (en) | 1982-02-02 |
IL57722A (en) | 1982-09-30 |
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