CA1304290C - Orthographic flow immunoassays and devices - Google Patents
Orthographic flow immunoassays and devicesInfo
- Publication number
- CA1304290C CA1304290C CA000557095A CA557095A CA1304290C CA 1304290 C CA1304290 C CA 1304290C CA 000557095 A CA000557095 A CA 000557095A CA 557095 A CA557095 A CA 557095A CA 1304290 C CA1304290 C CA 1304290C
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- Canada
- Prior art keywords
- matrix
- site
- sample medium
- plane
- directing
- 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.)
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
-
- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
-
- 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
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/97—Test strip or test slide
Abstract
ORTHOGRAPHIC FLOW IMMUNOASSAYS AND DEVICES
ABSTRACT OF THE DISCLOSURE
Methods and composition are provided for diag-nostic assays, where a sample traverses a bibulous matrix in a first plane and the flow path redirected into a second plane at a binding site, where a signal is created in relation to the presence of analyte.
Various protocols may be employed where components of a signal producing system may be combined with the sample, the matrix, or added at the binding site.
ABSTRACT OF THE DISCLOSURE
Methods and composition are provided for diag-nostic assays, where a sample traverses a bibulous matrix in a first plane and the flow path redirected into a second plane at a binding site, where a signal is created in relation to the presence of analyte.
Various protocols may be employed where components of a signal producing system may be combined with the sample, the matrix, or added at the binding site.
Description
~a~o 07RTHOGRAPHIC ~LO7~ IMMUNOASSAYS AN~ DEVICES
Dlagnostic a~ays have found expanding appli-cations in detecting a wide variety of drugs and other material~ of interest. There have been continuing e~forts to develop con~enient devices and protocols 0 which may be employed by untrained per~onnel, while providing for rapid, accurate re3ults. Such devices - require relatively simple equipment with minimal measurements and steps.
Many area~ o~ medicine, ~ood proces~ing, ln-dustrial proces~ing and rarming require the ability to detect the pre7~ence of a particular material. The need to mea7ure the drug or other sub~tance may be a~ a 7f 20 result of the abuse o~ drug3, the monitoring of thera-peutic do~age, the detection o~ a pathogen, the detec-tion of a di~cased ~tate, queh a~ neopla~ia, the detec-tion of contaminants or pollutants, or the concentra-- tion of a particular component, as illu~trative of the - 25 many situations which may be involved.
There ha~ been an increa~ing interest to re-~ove the requirement to measure a sub~tance in the ~linical laboratory and to mea~ure the substance at the site where the in~or~ation l~ to be u3ed. Thi~ may in-; 3 clude the doctor offiee, the home, the rarm, the ~ield or the proces~ing plant. In thi3 ~ituation, there are many restrictions on the nature Or the manner in wh~ch the determinatlon i3 to be earried out. For the most part, the devices must be 3$mple, rugged, and ea~ily ; ~5 handled. The protocols should also be ~imple, and involve a minimal number of measurements o~ sample and reagent, preferably zero, minimal handling and number ~' , ....
~3~4Z~
of reagent~, a~ well a~ a ~mall number of 3tep~. In addition, the reaults should be easy to read, particu-larly being vi~ually determined. In addition, there are other considerations such as preventing aerosoli-zation, providing reagent stability, and the like.
The design of such devices therefore requires efforts to optimize the various requirements, without unduly interfering with other requirements. Thus, as a practical matter, the device~ are only difficultly 10 conceived and reduced to practice.
Commercially available devices designed for home and doctor use include the ICON device provided by Hybritech, the Abbott device and the Pacific Biotech device. Patent disclosures of intere~t include U.S.
Patent No~. 4,435,504; 4,540,659; and references cited therein.
. Device~ and method~ are provided for deter~in-ing a .qubstance of intere~t, where orthographia flow i~
pro~ided, the ~ample medium migrating in a ~irst plane, followed by migration in 2 second plane, while any re-agentg are directed in the second plane. The device include~ one or more ports, a filter matrix, a b~nding pair member bound at an ob~ervation site, ~hich site ~erves a3 the site of redirection oP the flow path of the sample. One or more ~low directing materials may 3 be employed in con~unction with the matrix. In addi-tion, an ab~orbant i~ provided ~or absorbing the ~ample med1um and any additional media which are employed.
' .
. 35 ~L313 ~
In the drawings:
Fig. 1 1~ a ~ide elevation cross-sectional view of an embodiment of this invention;
Fig. 2 is an expanded per~pective view of a second embodiment of this invention;
Fig. 3 is a plan vLew of a rnatrix strip according to the inventSon;
Fig. 4 i~ a top view of a device provlding for determining a panel of a~says;
Fig. 5 is a cros~-sectional vlew of the device of Fig. 4 along~lines 5 5; and Flg. 6 i~ a batch device depicting three indi-vidual device~ ~oined together to carry out a plurality of assayi.
Method~ and device3 are provided which allow for a variety of protocols for the detect~on of ~ub-~tance~. Depending upon the nature of the ~ample, the particuiar analyte, and the con3truction and organ~-za~ion of the device, the protocol~ may vary.
The method involve~ directing a ~ample mediumin a fir~t direction through a bibulous matrix provid-ing for ~low by capillarity. One or more manipulative steps may oecur in the matrix. A binding æone ~ pro-vlded in the matrix proximal to the downstream ~nd ofthe matrix oppo~ite rrom ~he upstream end to which the sample medium 1~ added.
The ~ample medium pas~es through the binding 3 zone and is redireeted into a plane substantia}ly per-~ pendicular to the plane of the matrix, where the sample : medium and any liquid~ are absorbed, an absorbant serving ~ the receptacle for exce~ ~luid. A labeled reagent i~ employed, which label provides ~or a detec-table ~gnal. The level of signal in the binding zone, usually a color signal, Qerves to indicate the pre~ence and amount of the ~u~stance of intere~t, the analyte.
:
) The bibulous material or matrix may serve a wide variety of f~nctions. The matrix may serve to remove interfering materials ~or example, various matrices may serve to separate red blood cell~ and allow for the flow of serum free of the red blood cells and lytic contaminants. Variou~ materials may be removed by employin~ reciprocal binding members, which selectively remove a particular substance present in the sample medlum, including red blood cells or lytic ~o components thereof. Beside removal of contaminants or other ~ubstances which may interfere with the detection of the signal, the bibulous matrix may also serve as a source of reagents which may react wit'n the analyte or member of a signal producing ~y3tem. Thus, the matrix or solutions may be used for providing the various com-ponents of a ~ignal producing system involving the label.
In carrying out the assay, the sample may be used neat or may have been 3ub~ect to prior treatment.
The prior treatment may involve variou~ means of ~epa-ration, quch as centrifugation, for removing red blood cells, chromatography, heating, buffering, a~ well as ; the addition of various reagents. A choice may be made between providing for one or more reagent3 in the 9ample a~ contrasted with providi~g for the reagent~
bound to the matrix.
The nature of the 3ignal producing sy3tem, the costs of manufacturing, convenience, and other such considerations will determine whether the particular reagent i9 supplied bound to the matrix or is provided a~ a component to be added as part of the sample rnedium or a~ a sub3equent reagent. Reagents can there~ore be provided to be initially combined with the ~ample, as bound to the matrix, or added a~ a ~eparate medium to the binding zone.
In carrying out the a~say, the sample will be added proximal to one end of the matrix. Various tech-~3~ 9~
niques may be emplo~ed for directing the sample towardthe other en~ o~ tne matrix. During the traverse of the matrix, the ~ample may be subjected to interaction with variou~ reagents. These reagents ~ill be di3-cussed in discussing the signal producing system. Thesample traver~es the matrix undergoing the appropriate interactions with the reagents present on the matrix until it encounters the binding zone. The binding zone will involve a specific binding pair member which will bind to a reciprocal binding pair member, which may include the analyte, in relation to the amount of - analyte present in the sample. The binding zone is the reagent in which the signal is detected. The sample is then directed normal to the matrix to an absorbant which absorbs exce~s fluid.
The signal producing system may be varied widely, but will be subject to certain constraints.
The ~ignal producing system must provide a 3ignal which i9 related to the presence Or the analyte in the sample and in many situations will provide a semi-quantitative or quantitative qignal. In most situations, the signal producing sy3tem should pro~ide a signal which can be evaluated vi~ually, rather than u~ing instrumentation, although as appropriatel instrumentation may be em-ployed. Therefore, while fluorescence, magnetic flux, ultra-violet light absorption, or other non-visual signal may be employed, for the most part, the s~snal produclng 3ystem will provide a signal which iq the result of absorption of light in the visual range by a 3 dye. To this end, the signal producing system will usually employ an enzyme which catalyzes a reaction resulting in the ~ormation or de~truction of a dye ab-sorbing light in the visual range. In the~e instances, the enzyme will be conjugated to a member of a specific binding pair.
The specific binding pair will consist of ligand and receptor, where the terms are somewhat arbi-13~ 9~
trary, although generally understood as to theirmeaning. The receptor, ~or the most part, will be a macromolecule which binds to a speci~ic charge and ~patial conformation, having a high affinity for such S qpec1fic con~ormation a~ dlstlnct from molecules havin~
analogou~ but dl~ferent charge and spatial conforma-tion~. For the mo~t part, the receptor~ will be anti-bodies and therefore the assays are designated as im-munoa~ay~. However, other receptors may be employed, 10 particularly naturally occurring receptor3, which in-clude enzyme~, aectins, outer membrane protein~, 3uch a~ T-cell receptors, growth ~actor receptors, MHC
protein binding receptor3, etc., or blood proteins, ~ ~uch a~ thyroxine-binding globulin, avidin, and the - 15 l~e. As a special ca~e, the receptor may bë a nucleic acid, where the nucleic acid may bind to a prote~n or a complementary ~ingle ~tranded sequence.
The ligand may be any molecule ~or which re-ceptor~ are available or can be prepared. U3ual}y, the ligand will be an organic ~olecule o~ at lea~t about ~ 100 daltonQ (D) and may inYolve macromolecules, aggre-:~ gation~, cell~, viru~es, or the like. For the mo~t part, drug~ will generally be o~ about 125 to 2000 molecular weight, oligopeptide~ and protein will gen-25 erally ran~e from about 2 to 1000 kilodalSons (kD) and aggregate~ ~uch a~ organelles, ~embrane rragment~, viru~e~, or cell~ will be ~ub~tantially larger.
. The various analytes which may be detected in accordance ~ith the ~ub~ect in~ention are described in 30 U.S. Patent No. 4,261,968.
The enzyme con~ugate may take many dirferent ~orms, depending upon the particular protocol which i~
employed. The enzyme con~ugate may involYe an enzyme conjugated to ligand or receptor, where the ligand or receptor i9 part o~ the ~pecific bindin~ pair involving analyte or may be a receptor which binds to the con-I
3~3~
stant region of the immunoglobulin9 such as an antibodyto the F , S. aureus protein A, rheumatoid factor, or c the like. The enzyme may be a holoenzyme, apoenzyme, or enzyme fragment, where the fragment is capable o~
combining with a second fragment to provide a protein product having enzymatic activity.
Various combinations o~ reagents can be em-ployed. Constraints on the combinations of reagents in a particular medium and the timing of bringing the reagents together will include interactions between the reagents, for example, reaction o~ substrate with en-zyme, stability of the reagent, the time required ~or reaction, control of the amount of the reagent, and the like. For example, with an antigen analyte, one could provide for anti-antigen bound to the matrix at the binding site. One could then provide for enzyme-(anti-antigen) conjugate a~ a separate reagent, with enzyme substrate as a third reagent.
Alternatively, one could provide for an enzyme acceptor fragment at the binding site, which would ~erve a~ the receptor for the enzyme donor ~ragment.
See for example, U.S. Patent No. 4,378,428 and PCT/US85~02095 modified. The S peptide fragment o~
ribonuclea~e A or GNBr2 fragment of ~-galactosida~e may be conjugated with the analyte or a competitive frag-ment thereof. The ~ample could then be added to thereagent which would include the enzyme fragment conju-gate and the substrate for the enzyme. The matrix would include in a fir3t zone, anti-analyte, while the 3 binding zone will include the enzyme acceptor fragment.
Another alternative is employing the channel-ing reaction as is described in U.S. Patent No.
4,233,402. In this embodiment, a combination o~
enzyme~ ls used, where the product of one enzyme is the ~ubstrate of the other enzyme. In this embodiment, the sample could be combined with a second-enzyme-(anti-antigen) con~ugate and substrate for the first enzyme.
3~3~ 9~
The matrix would include anti-antigen and first enzyme in the binding region.
Various enzyme~ may be used in the signal pro-ducing system. The enzymes may be us~d individually or in combination, such as ~-galactosidase, malate dehy-drogenase, glucose-6-pho~phate dehydrogenase, acetyl-cholinesterase, alkaline phosphatase, glucose oxidase, horse radish peroxidase, urease, etc.
Substrates which may find use include: umbel-liferyl phosphate, salactosidyl fluorescein, tetra-methylbenzidine~ tetrazole salts, ABTS, or the like.
In addition, otner reagents rnay be bound to - the matrix, either diffu~ively or non-diffusively, such a~ receptors, enzymes, enzyme sub3trates, ligands, etc.
These various materials may be bound in the binding zone or as reagents upstream from the binding zone, where the diffusively bound reagents may migrate from the upstream region to the binding zone region.
Kits can be provided with the various reagents which may be used in conjunction with the device. The kits may include the various conjugate~ described above, ~uch as the enzyme-(anti-antigen) conjugate, the analyte-(enzyme fragment) conjugate, the anti-antigen and anti(anti-antigen)-conjugate, reagents such as bu~-fers, ~ub~trates ~or the enzyme, the matrix, or thelike.
The matrix may be any bibulou~ material which provideq for transport of an aqueous medium by capil-larity, as woll a~ binding of the desired reagent3. In addition, the matrix will de3irably minlmize the amount of non-specific binding in the binding zone and may provide for ancillary propertie~, such as separatlon o~
red blood cell~, removal of particulate matter, chro-matosraphic ~eparation, or the like.
~` 35 Various materials may be uced, both cellulosic and non-cellulosic, and these include glass fibers, particularly in the ran8e of about 0.2 to 5~, cellu-:~3(~29~
lose, nitrocellulose, paper, silica gel, etc. The matrix will generally be at lea~t about 2mm wide and usually not more than about 1cm wide, generally being at least about 0.5cm long and not more than about 5cm long, usu~lly not more than about 3cm long.
As already indicated, the matrix will have a binding zone which can be any convenient shape or for-mation and will serve as the site at which the signal is observed.
Various protocol~ may be employed for perform-ing the a~ay. ~A few protocols are provided a~ illu~-trative of different combinations of steps and reagents for carrying out the assay. These illustrative proto-col~ are not intended to be exhaustive, but rather i}lu~trative of particular embodiments.
In the first protocol to be described, the binding zone ha~ anti-antigen as a capture antibody.
The binding zone is positioned under a reagent addition and viewing port. Sample medium is added to the matrix at a po~ition distant and upstream from the binding zone and allowed to wick through the filter matrix while directed to the binding zone. The sample tra-ver3es the matrix to the binding zone in a fir~t direc-tion and i9 then directed through the binding zone to an absorbing layer in a direction normal to the first direction. Any analyte, in this ca~e antigen analyte, present in the 3ample will be captured in the binding zone by the capture antlbody. In ~ome application3 it may be bene~icial to filter the conjugate and~or other reagents through the orthogonal matrix before contact-ing the immunochemical surface, rather than adding them directly into the reagent addition (te~t) port.
The binding zone is then washed with a wash ~olution through the reagent addition port, ~ollowed by the addition o~ enzyme-(anti-antigen) conjugate through the reagent addition port. At this point, the reaction may be allowed to incubate ~ollowed by addition of a :iL3~
1 o wash solution to remove any non-specifically bound conjugate. The developing solution is then added con-taining all of the reagents necessary ~or the enzyme reaction to provide ~or a visual signal.
The individual wash solution~ are optional, depending upon the nature of the sample, the amount of interference that ma~ be expected from ~ample compo-nents, the amount of residual conjugate which may be retained in the binding zone, and the like. For 10 example, it may be found that the substrate solution suffices to remove any non-specifically bound enzyme conjugate, ~o as to 3ubstantially minimiæe the back-ground ~ignal. Also, it ma~ be found that the sample does not include any components which interfere ~ith 15 the binding reactions between the specific binding pair members, nor with the development of the signal.
A ~econd alternative protocol employs a zone upstream from the binding zone where the enzyme conju-gate i~ diffusively bound to the matrix. Addition of 20 the ~ample to the matrix re~ults in traversing the enzyme conjugate zone and carrying the enzyme conjugate with the sample to the binding zone. The binding zone may then be washed as described above, followed by addition of the development solution. As indicated 25 above, the wash solution iq optional, depending upon the nature of the sample, the amount of sample, and the c background ~ignal resulting ~rom non-speci~ically bound con~ugate.
In a third protocol, one may employ the chan-3 nelin~ ef~ect by having a fir~t enæyme bound to the matrix in the binding zone. A~ previously indicated, the ~irst enzyme produce~ a product which i~ the sub-~trate of the ~econd enzyma. The second enzyme pro-duce~ a product which provides for a visual signal. In 35 this protocol, the ~ample is added to a reagent con-taining second enzyme-(antl-antigen) conjugate, all of the nece~sary components o~ the enzyme reactions ~or both the first and the second enzymes, except for the product o~ the first enzyme, and any buffer~ or other reagents to optimize the development of the visual 3ig-nal. The sample may then be added to the matrix at a ~ite di3tant from the binding zone and allowed to tra-verse to the binding zone. The antigen may act as a bridge binding the ~econd enzyme to the binding zone by binding to anti-antigen in the binding zone. Rather than have the enzyme substrates together with the second enzyme-(anti-antigen) con~jugate, one may add them separately~through the port as preYiously de-~cribed after the sample has pas~ed through the binding zone.
Where a hapten i~ the analyte, rather than an antigen or receptor, the a~ay may be modified by hav-ing hapten pre3ent ln the binding zone. The sample may then be contacted with second enzyme-(anti-hapten). To the extent that the binding ~ites of the conjugate are filled with the hapten in the ~ample, the con~ugate will be unable to bind to the hapten present in the binding zone. Thus, the amount of ~econd enzyme present in the binding zone will be related to the amount of hapten in the sample.
An alternative technique i~ to use an enzyme fragment, which ~ay complex with another enzyme frag~
ment to provide for an enzymatlcally active protein.
For example, one may prepare a con~ugate of the S
peptide of ribonuclea~e At while binding the S-protein In the binding zone of the matrix. Anti-antigen may be 3 bound in a region upstream from the binding zone, which is kraver~ed by the ~ample. By combining the sample with the S-peptide con~ugate, the amount of S-peptide con~ugate which exits from the anti-anti~en region will be related to the amount of antisen in the sample. The S-antigen con~ugate which exits from the anti-antigen region will bind to the S-protein in the binding zone.
One may then add enzyme 3ub~trate to the binding zone :~3~
through the reagent addition port to detect any active enzyme.
In another protocol, one can provids for a series of regions in the matrix. A ~irst region would include antigen-(enzyme conjugate) di~fusively bound to the matrix. A second region would include anti-antigen. The binding region would be anti-enzyme. The sample mediu~ would be introduced upstream from the region~, 90 as to first traverse the enzyme-antigen conjugate which would be carried with the sample into the anti-antigen region, where antigen and enzyme-antigen conjugate would compete for the binding site~
o~ the anti-antigen. Any enzyme-antlgen conjugate which exited from the anti-antigen region would be cap-tured by the anti-enzyme pre~ent in the binding zone.
Once again, by adding a developer solution to the binding zone, the amount o~ signal produced would be related to the amount of analyte in the ~ample.
While the protocols have been de~cribed for hapten~ and antigens~ it i9 well known in the art to carry out analogou~ protocol~ with receptors. In the - - case of receptors, one would normally reverse the role of khe receptor w1th the antigen and vice ver~a.
Mean3 may be provided for directing the flow o~ the ~ample ~olution through the matrix lineariy and ; to the region of the binding zone, where the binding ; zone may as~ume a wide variety of configurations.
Thus, the path of the ~ample may be controlled as to direction and rate of flow.
Xn addition, the matrix may be provided with a control æone, which will be a~sociated with the binding zone, usually in close ~patial juxtaposition with the bindin& æone. The control region will provide for a signal which may be compared with the signal produced in the binding zone. The control region may provide for a fixed amount of enzyme bound to the matrix, which will produce a ~ignal level a~sociated with an amount of analyte in the range o~ interest. Alternatively, one could provide for an amount of anti-enzyme, which would bind enzyme conjugate at a predetermined level to provide a signal associated with an amount of sample in the range o~ interest. This approach would be particu-larly useful in sandwich assays, where the enzyme con-jugate i~ in exce~s over the amount to be bound to the ~urface in the binding zone. The control zone may be contiguou~ to the binding zone, separated from the binding zone, involved with forming pattern~ with the binding zone, or the like.
For some applications, it m~y be desirable to have a plurality of determinations carried out simul-taneously or consecutively with a ~Lngle unit. For ex-ample, one could provide for an apparatus having a hubwith a plurality o~ ~pokes providLng for the path of the sample. The ~ample would introduced at the hub and would radiate from the hub along the plurality o~
path~, each path could be treated with one or more dif-~erent reagent~, ~o as to allow for detection of di~-ferent analytes present in the sample. In this manner, a ~ingle sample could be analyzed for a family of ana-lytes, ~uch a~ dru~s of abuse~ pathogen~, or the like.
In other 3itutation~, it may be desirable to have a ~ingle apparatu incorporating a plurality of unit~, which may be used with the same or dif~erent samples and be carried out ~imultaneously. Thus, the various ~ample3 would be subjected to the same conditions.
This could be particularly u~eful if one wi~hes to employ a ~ingle control under the 3ame condition~ to which the ~ample i~ ~ubjected. Conveniently, the vari-ou~ unit~ could be joined together in a manner where they could be used either as a single unit or ~eparated one from the other to provide for individual indepen-dent units.
For further understanding of the invention,the figure~ will now be considered.
3~
In Fig. 1, a prototypic device (10) is de-picted. The device has a container (12) which includes a cover (14), which is di~posed below the top (16) of the container wall. The cover has a sample addition port (20) and a lens (22) for viewing. Below the cover (14) is a matrix (24), which matrix serves as the transport mechanism for transporting the sample by capillary action across the matrix in the direction of ~he arrows (26). The matrix (24) also serves to bind reagents, the reagents may remain bound to a particular region or may be~carried with the moving front o~ the ~ample medium across the matrix (24).
In the particular embodiment depicted in Fig. 1, the matrix contains enzyme-antigen conjugate (30) indicated a~ Ea, antibody to antigen (32) indi-cated a~ Aa as a zone downstream from the enzyme-antigen conjugate (30) and the binding zone (34) which includes antibody to enzyme (38~ depicted a~ Ae and ~ubstrate for the enzyme (36) indicated as S. Immedi-ately beneath the matrix (34) is water impermeablelayer (40) which serves to separate the matri~ (24) ~rom the absorbant (42). Various absorbants may be u~ed, such a~ cellulose, Filtrona, cotton, talc, silica - gel~ and the like. The absorbant may be a sponge-like material, powder, gel or other material which may ab-; . ~orb liquid ~rom the matrix (24) and serve a~ a recep-tacle for exces3 liquid. The absorbant (42) has pro-tuberance (44) which i9 in direct contact with the matrix (24) so as to allow for flow from the binding zone (34) into the absorbant layer (42). Plat~orm ~46) can be supported by springs (50~, if necessary, or other compres~ible structures in order to maintain the a~sembly under moderate pressure urging the assembly - toward cover (14).
In carrying out the assay, the ~ample medium would be introduced through port (20~, where the sample medium would be transported by capillary action in the 3L3~
direction indicated by arrow (26). As the sample medium passed the region containing the enzyme-antigen conjugate, the conjugate would be dissolved into the sample medium and transported with the sample medium front. The sample medium would then traverse the anti-antigen region where antigen in the ~ample medium would compete with antigen in the enzyme-antigen conjugate for the available binding ~ites of the anti-antigen.
Depending upon the amount of antigen in the sample, enzyme-antigen would exit the anti-antigen region and continue to the binding region (34). Any enzyme-antigen conjugate in the medium would be captured by the anti-enzyme, which is non-diffusively bound. The substrate would dissolve into the medium and react with the enzyme present in the binding region producing a product which would ~trongly bind to the matrix (24).
The product would be darkly colored, for example, black, and would produce a dark spot oYer a predeter-mined time period, where the ab~orption of the spot would be related to the amount of antigen in the sample. One would view the spot through lens (22), ~o as to get a qualitative determination of the presence and amount of antigen in the sample.
In Fig. 2 another embodiment is depicted.
25 Thi9 device (60) has a container (62). The container - (62) has top closure (64). Top closure (64) ha~ port (66) into which sample receptacle (70) feed~ a Yample.
Top closure (64) has a second port (72) into which reagent and wash solution receptaole (74) feed~ the 3 appropriate liquid media. Pre~sed against top closure (64) i~ filter matrix (76) with binding region (80).
The filter matrix (76) is incorporated into a thin flow direction separator (82). Such qeparators can be illustrated by the following examples.
1. The separator could have the filter matrix (76) inserted into a raised lip (84) which contains the filter matrix and, effectively, direots the flow of ~ample ~luid to the binding reglon (80).
The ~eparator has orifice (86~ through which the sample flows from the binding region through a unidirectional ~low ~ilm (89). The ab30rbant matrix t90) which contact~ the unidirectional flow ~ilm (89~ receives the excess fluid and withdraw~ the fluid from the filter matrix (76).
Dlagnostic a~ays have found expanding appli-cations in detecting a wide variety of drugs and other material~ of interest. There have been continuing e~forts to develop con~enient devices and protocols 0 which may be employed by untrained per~onnel, while providing for rapid, accurate re3ults. Such devices - require relatively simple equipment with minimal measurements and steps.
Many area~ o~ medicine, ~ood proces~ing, ln-dustrial proces~ing and rarming require the ability to detect the pre7~ence of a particular material. The need to mea7ure the drug or other sub~tance may be a~ a 7f 20 result of the abuse o~ drug3, the monitoring of thera-peutic do~age, the detection o~ a pathogen, the detec-tion of a di~cased ~tate, queh a~ neopla~ia, the detec-tion of contaminants or pollutants, or the concentra-- tion of a particular component, as illu~trative of the - 25 many situations which may be involved.
There ha~ been an increa~ing interest to re-~ove the requirement to measure a sub~tance in the ~linical laboratory and to mea~ure the substance at the site where the in~or~ation l~ to be u3ed. Thi~ may in-; 3 clude the doctor offiee, the home, the rarm, the ~ield or the proces~ing plant. In thi3 ~ituation, there are many restrictions on the nature Or the manner in wh~ch the determinatlon i3 to be earried out. For the most part, the devices must be 3$mple, rugged, and ea~ily ; ~5 handled. The protocols should also be ~imple, and involve a minimal number of measurements o~ sample and reagent, preferably zero, minimal handling and number ~' , ....
~3~4Z~
of reagent~, a~ well a~ a ~mall number of 3tep~. In addition, the reaults should be easy to read, particu-larly being vi~ually determined. In addition, there are other considerations such as preventing aerosoli-zation, providing reagent stability, and the like.
The design of such devices therefore requires efforts to optimize the various requirements, without unduly interfering with other requirements. Thus, as a practical matter, the device~ are only difficultly 10 conceived and reduced to practice.
Commercially available devices designed for home and doctor use include the ICON device provided by Hybritech, the Abbott device and the Pacific Biotech device. Patent disclosures of intere~t include U.S.
Patent No~. 4,435,504; 4,540,659; and references cited therein.
. Device~ and method~ are provided for deter~in-ing a .qubstance of intere~t, where orthographia flow i~
pro~ided, the ~ample medium migrating in a ~irst plane, followed by migration in 2 second plane, while any re-agentg are directed in the second plane. The device include~ one or more ports, a filter matrix, a b~nding pair member bound at an ob~ervation site, ~hich site ~erves a3 the site of redirection oP the flow path of the sample. One or more ~low directing materials may 3 be employed in con~unction with the matrix. In addi-tion, an ab~orbant i~ provided ~or absorbing the ~ample med1um and any additional media which are employed.
' .
. 35 ~L313 ~
In the drawings:
Fig. 1 1~ a ~ide elevation cross-sectional view of an embodiment of this invention;
Fig. 2 is an expanded per~pective view of a second embodiment of this invention;
Fig. 3 is a plan vLew of a rnatrix strip according to the inventSon;
Fig. 4 i~ a top view of a device provlding for determining a panel of a~says;
Fig. 5 is a cros~-sectional vlew of the device of Fig. 4 along~lines 5 5; and Flg. 6 i~ a batch device depicting three indi-vidual device~ ~oined together to carry out a plurality of assayi.
Method~ and device3 are provided which allow for a variety of protocols for the detect~on of ~ub-~tance~. Depending upon the nature of the ~ample, the particuiar analyte, and the con3truction and organ~-za~ion of the device, the protocol~ may vary.
The method involve~ directing a ~ample mediumin a fir~t direction through a bibulous matrix provid-ing for ~low by capillarity. One or more manipulative steps may oecur in the matrix. A binding æone ~ pro-vlded in the matrix proximal to the downstream ~nd ofthe matrix oppo~ite rrom ~he upstream end to which the sample medium 1~ added.
The ~ample medium pas~es through the binding 3 zone and is redireeted into a plane substantia}ly per-~ pendicular to the plane of the matrix, where the sample : medium and any liquid~ are absorbed, an absorbant serving ~ the receptacle for exce~ ~luid. A labeled reagent i~ employed, which label provides ~or a detec-table ~gnal. The level of signal in the binding zone, usually a color signal, Qerves to indicate the pre~ence and amount of the ~u~stance of intere~t, the analyte.
:
) The bibulous material or matrix may serve a wide variety of f~nctions. The matrix may serve to remove interfering materials ~or example, various matrices may serve to separate red blood cell~ and allow for the flow of serum free of the red blood cells and lytic contaminants. Variou~ materials may be removed by employin~ reciprocal binding members, which selectively remove a particular substance present in the sample medlum, including red blood cells or lytic ~o components thereof. Beside removal of contaminants or other ~ubstances which may interfere with the detection of the signal, the bibulous matrix may also serve as a source of reagents which may react wit'n the analyte or member of a signal producing ~y3tem. Thus, the matrix or solutions may be used for providing the various com-ponents of a ~ignal producing system involving the label.
In carrying out the assay, the sample may be used neat or may have been 3ub~ect to prior treatment.
The prior treatment may involve variou~ means of ~epa-ration, quch as centrifugation, for removing red blood cells, chromatography, heating, buffering, a~ well as ; the addition of various reagents. A choice may be made between providing for one or more reagent3 in the 9ample a~ contrasted with providi~g for the reagent~
bound to the matrix.
The nature of the 3ignal producing sy3tem, the costs of manufacturing, convenience, and other such considerations will determine whether the particular reagent i9 supplied bound to the matrix or is provided a~ a component to be added as part of the sample rnedium or a~ a sub3equent reagent. Reagents can there~ore be provided to be initially combined with the ~ample, as bound to the matrix, or added a~ a ~eparate medium to the binding zone.
In carrying out the a~say, the sample will be added proximal to one end of the matrix. Various tech-~3~ 9~
niques may be emplo~ed for directing the sample towardthe other en~ o~ tne matrix. During the traverse of the matrix, the ~ample may be subjected to interaction with variou~ reagents. These reagents ~ill be di3-cussed in discussing the signal producing system. Thesample traver~es the matrix undergoing the appropriate interactions with the reagents present on the matrix until it encounters the binding zone. The binding zone will involve a specific binding pair member which will bind to a reciprocal binding pair member, which may include the analyte, in relation to the amount of - analyte present in the sample. The binding zone is the reagent in which the signal is detected. The sample is then directed normal to the matrix to an absorbant which absorbs exce~s fluid.
The signal producing system may be varied widely, but will be subject to certain constraints.
The ~ignal producing system must provide a 3ignal which i9 related to the presence Or the analyte in the sample and in many situations will provide a semi-quantitative or quantitative qignal. In most situations, the signal producing sy3tem should pro~ide a signal which can be evaluated vi~ually, rather than u~ing instrumentation, although as appropriatel instrumentation may be em-ployed. Therefore, while fluorescence, magnetic flux, ultra-violet light absorption, or other non-visual signal may be employed, for the most part, the s~snal produclng 3ystem will provide a signal which iq the result of absorption of light in the visual range by a 3 dye. To this end, the signal producing system will usually employ an enzyme which catalyzes a reaction resulting in the ~ormation or de~truction of a dye ab-sorbing light in the visual range. In the~e instances, the enzyme will be conjugated to a member of a specific binding pair.
The specific binding pair will consist of ligand and receptor, where the terms are somewhat arbi-13~ 9~
trary, although generally understood as to theirmeaning. The receptor, ~or the most part, will be a macromolecule which binds to a speci~ic charge and ~patial conformation, having a high affinity for such S qpec1fic con~ormation a~ dlstlnct from molecules havin~
analogou~ but dl~ferent charge and spatial conforma-tion~. For the mo~t part, the receptor~ will be anti-bodies and therefore the assays are designated as im-munoa~ay~. However, other receptors may be employed, 10 particularly naturally occurring receptor3, which in-clude enzyme~, aectins, outer membrane protein~, 3uch a~ T-cell receptors, growth ~actor receptors, MHC
protein binding receptor3, etc., or blood proteins, ~ ~uch a~ thyroxine-binding globulin, avidin, and the - 15 l~e. As a special ca~e, the receptor may bë a nucleic acid, where the nucleic acid may bind to a prote~n or a complementary ~ingle ~tranded sequence.
The ligand may be any molecule ~or which re-ceptor~ are available or can be prepared. U3ual}y, the ligand will be an organic ~olecule o~ at lea~t about ~ 100 daltonQ (D) and may inYolve macromolecules, aggre-:~ gation~, cell~, viru~es, or the like. For the mo~t part, drug~ will generally be o~ about 125 to 2000 molecular weight, oligopeptide~ and protein will gen-25 erally ran~e from about 2 to 1000 kilodalSons (kD) and aggregate~ ~uch a~ organelles, ~embrane rragment~, viru~e~, or cell~ will be ~ub~tantially larger.
. The various analytes which may be detected in accordance ~ith the ~ub~ect in~ention are described in 30 U.S. Patent No. 4,261,968.
The enzyme con~ugate may take many dirferent ~orms, depending upon the particular protocol which i~
employed. The enzyme con~ugate may involYe an enzyme conjugated to ligand or receptor, where the ligand or receptor i9 part o~ the ~pecific bindin~ pair involving analyte or may be a receptor which binds to the con-I
3~3~
stant region of the immunoglobulin9 such as an antibodyto the F , S. aureus protein A, rheumatoid factor, or c the like. The enzyme may be a holoenzyme, apoenzyme, or enzyme fragment, where the fragment is capable o~
combining with a second fragment to provide a protein product having enzymatic activity.
Various combinations o~ reagents can be em-ployed. Constraints on the combinations of reagents in a particular medium and the timing of bringing the reagents together will include interactions between the reagents, for example, reaction o~ substrate with en-zyme, stability of the reagent, the time required ~or reaction, control of the amount of the reagent, and the like. For example, with an antigen analyte, one could provide for anti-antigen bound to the matrix at the binding site. One could then provide for enzyme-(anti-antigen) conjugate a~ a separate reagent, with enzyme substrate as a third reagent.
Alternatively, one could provide for an enzyme acceptor fragment at the binding site, which would ~erve a~ the receptor for the enzyme donor ~ragment.
See for example, U.S. Patent No. 4,378,428 and PCT/US85~02095 modified. The S peptide fragment o~
ribonuclea~e A or GNBr2 fragment of ~-galactosida~e may be conjugated with the analyte or a competitive frag-ment thereof. The ~ample could then be added to thereagent which would include the enzyme fragment conju-gate and the substrate for the enzyme. The matrix would include in a fir3t zone, anti-analyte, while the 3 binding zone will include the enzyme acceptor fragment.
Another alternative is employing the channel-ing reaction as is described in U.S. Patent No.
4,233,402. In this embodiment, a combination o~
enzyme~ ls used, where the product of one enzyme is the ~ubstrate of the other enzyme. In this embodiment, the sample could be combined with a second-enzyme-(anti-antigen) con~ugate and substrate for the first enzyme.
3~3~ 9~
The matrix would include anti-antigen and first enzyme in the binding region.
Various enzyme~ may be used in the signal pro-ducing system. The enzymes may be us~d individually or in combination, such as ~-galactosidase, malate dehy-drogenase, glucose-6-pho~phate dehydrogenase, acetyl-cholinesterase, alkaline phosphatase, glucose oxidase, horse radish peroxidase, urease, etc.
Substrates which may find use include: umbel-liferyl phosphate, salactosidyl fluorescein, tetra-methylbenzidine~ tetrazole salts, ABTS, or the like.
In addition, otner reagents rnay be bound to - the matrix, either diffu~ively or non-diffusively, such a~ receptors, enzymes, enzyme sub3trates, ligands, etc.
These various materials may be bound in the binding zone or as reagents upstream from the binding zone, where the diffusively bound reagents may migrate from the upstream region to the binding zone region.
Kits can be provided with the various reagents which may be used in conjunction with the device. The kits may include the various conjugate~ described above, ~uch as the enzyme-(anti-antigen) conjugate, the analyte-(enzyme fragment) conjugate, the anti-antigen and anti(anti-antigen)-conjugate, reagents such as bu~-fers, ~ub~trates ~or the enzyme, the matrix, or thelike.
The matrix may be any bibulou~ material which provideq for transport of an aqueous medium by capil-larity, as woll a~ binding of the desired reagent3. In addition, the matrix will de3irably minlmize the amount of non-specific binding in the binding zone and may provide for ancillary propertie~, such as separatlon o~
red blood cell~, removal of particulate matter, chro-matosraphic ~eparation, or the like.
~` 35 Various materials may be uced, both cellulosic and non-cellulosic, and these include glass fibers, particularly in the ran8e of about 0.2 to 5~, cellu-:~3(~29~
lose, nitrocellulose, paper, silica gel, etc. The matrix will generally be at lea~t about 2mm wide and usually not more than about 1cm wide, generally being at least about 0.5cm long and not more than about 5cm long, usu~lly not more than about 3cm long.
As already indicated, the matrix will have a binding zone which can be any convenient shape or for-mation and will serve as the site at which the signal is observed.
Various protocol~ may be employed for perform-ing the a~ay. ~A few protocols are provided a~ illu~-trative of different combinations of steps and reagents for carrying out the assay. These illustrative proto-col~ are not intended to be exhaustive, but rather i}lu~trative of particular embodiments.
In the first protocol to be described, the binding zone ha~ anti-antigen as a capture antibody.
The binding zone is positioned under a reagent addition and viewing port. Sample medium is added to the matrix at a po~ition distant and upstream from the binding zone and allowed to wick through the filter matrix while directed to the binding zone. The sample tra-ver3es the matrix to the binding zone in a fir~t direc-tion and i9 then directed through the binding zone to an absorbing layer in a direction normal to the first direction. Any analyte, in this ca~e antigen analyte, present in the 3ample will be captured in the binding zone by the capture antlbody. In ~ome application3 it may be bene~icial to filter the conjugate and~or other reagents through the orthogonal matrix before contact-ing the immunochemical surface, rather than adding them directly into the reagent addition (te~t) port.
The binding zone is then washed with a wash ~olution through the reagent addition port, ~ollowed by the addition o~ enzyme-(anti-antigen) conjugate through the reagent addition port. At this point, the reaction may be allowed to incubate ~ollowed by addition of a :iL3~
1 o wash solution to remove any non-specifically bound conjugate. The developing solution is then added con-taining all of the reagents necessary ~or the enzyme reaction to provide ~or a visual signal.
The individual wash solution~ are optional, depending upon the nature of the sample, the amount of interference that ma~ be expected from ~ample compo-nents, the amount of residual conjugate which may be retained in the binding zone, and the like. For 10 example, it may be found that the substrate solution suffices to remove any non-specifically bound enzyme conjugate, ~o as to 3ubstantially minimiæe the back-ground ~ignal. Also, it ma~ be found that the sample does not include any components which interfere ~ith 15 the binding reactions between the specific binding pair members, nor with the development of the signal.
A ~econd alternative protocol employs a zone upstream from the binding zone where the enzyme conju-gate i~ diffusively bound to the matrix. Addition of 20 the ~ample to the matrix re~ults in traversing the enzyme conjugate zone and carrying the enzyme conjugate with the sample to the binding zone. The binding zone may then be washed as described above, followed by addition of the development solution. As indicated 25 above, the wash solution iq optional, depending upon the nature of the sample, the amount of sample, and the c background ~ignal resulting ~rom non-speci~ically bound con~ugate.
In a third protocol, one may employ the chan-3 nelin~ ef~ect by having a fir~t enæyme bound to the matrix in the binding zone. A~ previously indicated, the ~irst enzyme produce~ a product which i~ the sub-~trate of the ~econd enzyma. The second enzyme pro-duce~ a product which provides for a visual signal. In 35 this protocol, the ~ample is added to a reagent con-taining second enzyme-(antl-antigen) conjugate, all of the nece~sary components o~ the enzyme reactions ~or both the first and the second enzymes, except for the product o~ the first enzyme, and any buffer~ or other reagents to optimize the development of the visual 3ig-nal. The sample may then be added to the matrix at a ~ite di3tant from the binding zone and allowed to tra-verse to the binding zone. The antigen may act as a bridge binding the ~econd enzyme to the binding zone by binding to anti-antigen in the binding zone. Rather than have the enzyme substrates together with the second enzyme-(anti-antigen) con~jugate, one may add them separately~through the port as preYiously de-~cribed after the sample has pas~ed through the binding zone.
Where a hapten i~ the analyte, rather than an antigen or receptor, the a~ay may be modified by hav-ing hapten pre3ent ln the binding zone. The sample may then be contacted with second enzyme-(anti-hapten). To the extent that the binding ~ites of the conjugate are filled with the hapten in the ~ample, the con~ugate will be unable to bind to the hapten present in the binding zone. Thus, the amount of ~econd enzyme present in the binding zone will be related to the amount of hapten in the sample.
An alternative technique i~ to use an enzyme fragment, which ~ay complex with another enzyme frag~
ment to provide for an enzymatlcally active protein.
For example, one may prepare a con~ugate of the S
peptide of ribonuclea~e At while binding the S-protein In the binding zone of the matrix. Anti-antigen may be 3 bound in a region upstream from the binding zone, which is kraver~ed by the ~ample. By combining the sample with the S-peptide con~ugate, the amount of S-peptide con~ugate which exits from the anti-anti~en region will be related to the amount of antisen in the sample. The S-antigen con~ugate which exits from the anti-antigen region will bind to the S-protein in the binding zone.
One may then add enzyme 3ub~trate to the binding zone :~3~
through the reagent addition port to detect any active enzyme.
In another protocol, one can provids for a series of regions in the matrix. A ~irst region would include antigen-(enzyme conjugate) di~fusively bound to the matrix. A second region would include anti-antigen. The binding region would be anti-enzyme. The sample mediu~ would be introduced upstream from the region~, 90 as to first traverse the enzyme-antigen conjugate which would be carried with the sample into the anti-antigen region, where antigen and enzyme-antigen conjugate would compete for the binding site~
o~ the anti-antigen. Any enzyme-antlgen conjugate which exited from the anti-antigen region would be cap-tured by the anti-enzyme pre~ent in the binding zone.
Once again, by adding a developer solution to the binding zone, the amount o~ signal produced would be related to the amount of analyte in the ~ample.
While the protocols have been de~cribed for hapten~ and antigens~ it i9 well known in the art to carry out analogou~ protocol~ with receptors. In the - - case of receptors, one would normally reverse the role of khe receptor w1th the antigen and vice ver~a.
Mean3 may be provided for directing the flow o~ the ~ample ~olution through the matrix lineariy and ; to the region of the binding zone, where the binding ; zone may as~ume a wide variety of configurations.
Thus, the path of the ~ample may be controlled as to direction and rate of flow.
Xn addition, the matrix may be provided with a control æone, which will be a~sociated with the binding zone, usually in close ~patial juxtaposition with the bindin& æone. The control region will provide for a signal which may be compared with the signal produced in the binding zone. The control region may provide for a fixed amount of enzyme bound to the matrix, which will produce a ~ignal level a~sociated with an amount of analyte in the range o~ interest. Alternatively, one could provide for an amount of anti-enzyme, which would bind enzyme conjugate at a predetermined level to provide a signal associated with an amount of sample in the range o~ interest. This approach would be particu-larly useful in sandwich assays, where the enzyme con-jugate i~ in exce~s over the amount to be bound to the ~urface in the binding zone. The control zone may be contiguou~ to the binding zone, separated from the binding zone, involved with forming pattern~ with the binding zone, or the like.
For some applications, it m~y be desirable to have a plurality of determinations carried out simul-taneously or consecutively with a ~Lngle unit. For ex-ample, one could provide for an apparatus having a hubwith a plurality o~ ~pokes providLng for the path of the sample. The ~ample would introduced at the hub and would radiate from the hub along the plurality o~
path~, each path could be treated with one or more dif-~erent reagent~, ~o as to allow for detection of di~-ferent analytes present in the sample. In this manner, a ~ingle sample could be analyzed for a family of ana-lytes, ~uch a~ dru~s of abuse~ pathogen~, or the like.
In other 3itutation~, it may be desirable to have a ~ingle apparatu incorporating a plurality of unit~, which may be used with the same or dif~erent samples and be carried out ~imultaneously. Thus, the various ~ample3 would be subjected to the same conditions.
This could be particularly u~eful if one wi~hes to employ a ~ingle control under the 3ame condition~ to which the ~ample i~ ~ubjected. Conveniently, the vari-ou~ unit~ could be joined together in a manner where they could be used either as a single unit or ~eparated one from the other to provide for individual indepen-dent units.
For further understanding of the invention,the figure~ will now be considered.
3~
In Fig. 1, a prototypic device (10) is de-picted. The device has a container (12) which includes a cover (14), which is di~posed below the top (16) of the container wall. The cover has a sample addition port (20) and a lens (22) for viewing. Below the cover (14) is a matrix (24), which matrix serves as the transport mechanism for transporting the sample by capillary action across the matrix in the direction of ~he arrows (26). The matrix (24) also serves to bind reagents, the reagents may remain bound to a particular region or may be~carried with the moving front o~ the ~ample medium across the matrix (24).
In the particular embodiment depicted in Fig. 1, the matrix contains enzyme-antigen conjugate (30) indicated a~ Ea, antibody to antigen (32) indi-cated a~ Aa as a zone downstream from the enzyme-antigen conjugate (30) and the binding zone (34) which includes antibody to enzyme (38~ depicted a~ Ae and ~ubstrate for the enzyme (36) indicated as S. Immedi-ately beneath the matrix (34) is water impermeablelayer (40) which serves to separate the matri~ (24) ~rom the absorbant (42). Various absorbants may be u~ed, such a~ cellulose, Filtrona, cotton, talc, silica - gel~ and the like. The absorbant may be a sponge-like material, powder, gel or other material which may ab-; . ~orb liquid ~rom the matrix (24) and serve a~ a recep-tacle for exces3 liquid. The absorbant (42) has pro-tuberance (44) which i9 in direct contact with the matrix (24) so as to allow for flow from the binding zone (34) into the absorbant layer (42). Plat~orm ~46) can be supported by springs (50~, if necessary, or other compres~ible structures in order to maintain the a~sembly under moderate pressure urging the assembly - toward cover (14).
In carrying out the assay, the ~ample medium would be introduced through port (20~, where the sample medium would be transported by capillary action in the 3L3~
direction indicated by arrow (26). As the sample medium passed the region containing the enzyme-antigen conjugate, the conjugate would be dissolved into the sample medium and transported with the sample medium front. The sample medium would then traverse the anti-antigen region where antigen in the ~ample medium would compete with antigen in the enzyme-antigen conjugate for the available binding ~ites of the anti-antigen.
Depending upon the amount of antigen in the sample, enzyme-antigen would exit the anti-antigen region and continue to the binding region (34). Any enzyme-antigen conjugate in the medium would be captured by the anti-enzyme, which is non-diffusively bound. The substrate would dissolve into the medium and react with the enzyme present in the binding region producing a product which would ~trongly bind to the matrix (24).
The product would be darkly colored, for example, black, and would produce a dark spot oYer a predeter-mined time period, where the ab~orption of the spot would be related to the amount of antigen in the sample. One would view the spot through lens (22), ~o as to get a qualitative determination of the presence and amount of antigen in the sample.
In Fig. 2 another embodiment is depicted.
25 Thi9 device (60) has a container (62). The container - (62) has top closure (64). Top closure (64) ha~ port (66) into which sample receptacle (70) feed~ a Yample.
Top closure (64) has a second port (72) into which reagent and wash solution receptaole (74) feed~ the 3 appropriate liquid media. Pre~sed against top closure (64) i~ filter matrix (76) with binding region (80).
The filter matrix (76) is incorporated into a thin flow direction separator (82). Such qeparators can be illustrated by the following examples.
1. The separator could have the filter matrix (76) inserted into a raised lip (84) which contains the filter matrix and, effectively, direots the flow of ~ample ~luid to the binding reglon (80).
The ~eparator has orifice (86~ through which the sample flows from the binding region through a unidirectional ~low ~ilm (89). The ab30rbant matrix t90) which contact~ the unidirectional flow ~ilm (89~ receives the excess fluid and withdraw~ the fluid from the filter matrix (76).
2. The separator, al~ernatively 7 could be con~tructed from three laminated pla~tic sheet3, with the middle layer cut out in a ~hape to match the filter matrix (76) (not ~hown~. The top ~heet contain~ the ~ample addition port ~equivalent to 66~ and the top te3t port (equivalent to 72). The bottom ~heet contain~ the exlt test port (equivalent So 86) and 15 makes intimate contact with the unidirectional flow film (89). the unidirectional flow film in turn makes lntimate contact with the ab~orbant matrix (90).
An as~ay may be carried out, ~or example, by combining 3ample containin~ analyte, ~or example antige~, with enzyme-antigen con~ugate and adding it to reoeptacle (70). The sample then pa~s~ through port ~66) and proceed~ in the direction of arrow~ (92) into the filter matrix (76), where it i~ directed by separator (82) toward the binding region (80). The 2~ ~ample medium pa~es thruugh the binding region (80) and proceed~ in the direction o~ arrow ~94) to ab~orbant (90), where the liquid ~pread~ out as : indicated by arrow~ ~9~). Antibody in the binding region (80) captures enzyme-antigen conjugate in proportion to the amount o~ antigen pre~ent in the 3ample. After the sample ha~ been exhau~ted, ~o that no further ~ample r~main~ ~n the ample receptacle (70), a wa~h 301ution may be added to receptacle (74) to wa~h away any non-3pecifically bound enzyme-antigen con~ugate. ~hen the receptacle (74) iq emp~y, a substrate ~olution may be added to flll the receptacle and the 3ub~trate ~olution allowed to traver~e through the binding region (80) to be absorbed by absorbant (90). The binding region (80) may then be viewed through port (72), where the presence of color is indicative o~ the presence of analyte.
Alternatively, rather than combine the antigen with an enzyme-antigen conjugate, one could ~irst allow the ~ample to traverse the matrix and pass through the binding region, filling up a proportional number of binding sites. One could then add enzyme-antibody conjugate which would bind to antigen captured by antibody in the binding region. The procedure would then follow as described above.
In Fig. 3, i~ depicted an alternate form of a matrix (100). In this matrix, antibodies to red blood cells (102) are bound to the matrLx indicated at ArbC.
Where blood is employed as a sample, the antibodies (102) will ~erve to remove the red blood cell3 ~rom the kraveling sample medium, so that any red blood cell~
will not interfere with the detection of color in the binding region (104). The binding region (104) i~
indicated a~ a circle, but any design may be employed, ~uch a~ a bar, cros3, triangle, or the like. Surround-ing the binding region (104) is control region (106~.
Control region (105) ha3 a predetermined amount of enzyme (110) indicated as E. ~y pefor~ing the as3ay as de~cribed above, the amount of enzyme conjugate bound to the binding region will be related to the amount o~
antigen in the medium. B~ comparing the color produced in the binding region with the color produced in the control region, one can determine whether the a~ount of antigen in the sample exceeds a predetermined level.
This will be particularly useful, where the result i3 either positive or negatiYe~ depending upon whether the - analyte is above a predetermined level.
Figs. 4 and 5 depict a device which may be used for determining a panel of drug3 ~o that a single ~ample may be treated in a variety of ways to give a ~3Q~ 3~3 variety of result~. Panel device (120) is a filter top plate (122) under which appears a plurality of matrixes (124). The individual matrixes may be of the qame or different length3, as required. The matrixes are joined at a central hub region (126). The hub region contacts each of the filter matrices (124) and feeds the ~ample into each matrix. Inlet (128) connects well (130) to the hub (126). The well (130) allows for a measured amount of sample to be added to the well which will then be absorbed into the hub region (126) and be tran~mitted evenl~y to the various filter matrices (124). A plurality o~ reagent addition ports (132) are provided which intimately contact the ends of each matrix (124). Ports (132) are surrounded by a well (134) which allows for the introduction of various reagents through the port. The matrix i9 retained in plate (136), where plate (136) has been cut out to house matrices (124) and hub (126) and further act as a divider between the matrices (124). The plate (136) i9 ~upported by 3eparator plate (138) which intimately contact~ a unidirectional ~low membrane (139), which in turn intimately contacts absorbant material (140). The absorbed fluid components are retained in housing : (144).
In Fig. 6, multiple unit ~150) has a plurality of devices (152) joined together at broken line (154) indicating the presence of a fracture line, allowlng for separation of the individual unit~ de~ired. As in the previous unit~, there is a sample well (156) in the port (158) and a reagent port (160) with well (162) where the ~ilter matrix (164) is indicated by the broken lines.
A~ i~ evident from the above description, the ~ubject device~ and protocols provide for a large num-ber of advantages. As contrasted to other deviceswhich are commercially available, in accordance with the subject invention the sample may be pre-treated ~L3~i~2910 without the necessity for physical removal of a pre-treatment filter, thus avoiding aerosolization of in-fectious or potentially infectious samples. Filtration of sample parallel to the thin dimension of the filter matrix as opposed to perpendicular to the filter matrix as is currently being done or has been depicted in the literature allows for the physical separation of blood cells usins selected materials, such as glass fLber filter~ or filter matrix containing binding ligands, which inhibit the migration of the red blood cells.
The sample may be filtered through a much longer linear dimension of filter material prior to arriving at the binding regionq The ~ilter matrix can be utilized to allow administration, mixing and reaction of immuno-logical components or other pre-treatment components without physical manipulation of these materials.
Thus, user steps may be eliminated from the assay pro-tocol for greater convenience and test reliability. In the subject devices, the matrlx serves as a u~e~ul medium for 3tabilizing dried immunological and pre-treatment reagent~ in the assay device. Since such dried materLal3 are typically more stable than materi-als provided in liquid form, the device~ are more amenable to long ~helf life or 3torage at elevated temperatures. This is a particularly lmportant feature for an over-the-counter or con~umer-oriented product or ior general rield u~e where refrigeration is imprac-tical. In addition, the nature of the ~ilter may be varied in order to allow modulation of the migration time across the matrix to the binding region. This provides a means ior controlling the time of the immu-nological raactions or pre-treatment reactions which czn be provided for with the iilter matrix.
By providing for a long path ~low of fluids, one can allow the sample to traver~e a filter matrix over a relatively lon~ path, or providing for a short path to the binding region for reagents and wash solu-~3~42t3~
2~
tions, where the reag~nts may be provided at a substan-tially constant concentration to the binding region.
The long path of the sample provides for removal of in-terfering materials in an efficient manner. The short path for the wash solutions and reagent provides econo-mies in time, improved control of the contact between reagents, such as conjugates and developer solutions, and may reduce the amount of solution required for ob-taining the desired result.
Multiple test devices allow for performing a panel of related~tests on a single specimen or a series ~batch) of the same tests on a group of different sample~. Multiple test devices provide cost economics in decreasing the cost per reportable result.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of under~tanding, it will be obvious that certain changes and modifications may be practiced within the scope o~ the appended claims-~5 .
3o
An as~ay may be carried out, ~or example, by combining 3ample containin~ analyte, ~or example antige~, with enzyme-antigen con~ugate and adding it to reoeptacle (70). The sample then pa~s~ through port ~66) and proceed~ in the direction of arrow~ (92) into the filter matrix (76), where it i~ directed by separator (82) toward the binding region (80). The 2~ ~ample medium pa~es thruugh the binding region (80) and proceed~ in the direction o~ arrow ~94) to ab~orbant (90), where the liquid ~pread~ out as : indicated by arrow~ ~9~). Antibody in the binding region (80) captures enzyme-antigen conjugate in proportion to the amount o~ antigen pre~ent in the 3ample. After the sample ha~ been exhau~ted, ~o that no further ~ample r~main~ ~n the ample receptacle (70), a wa~h 301ution may be added to receptacle (74) to wa~h away any non-3pecifically bound enzyme-antigen con~ugate. ~hen the receptacle (74) iq emp~y, a substrate ~olution may be added to flll the receptacle and the 3ub~trate ~olution allowed to traver~e through the binding region (80) to be absorbed by absorbant (90). The binding region (80) may then be viewed through port (72), where the presence of color is indicative o~ the presence of analyte.
Alternatively, rather than combine the antigen with an enzyme-antigen conjugate, one could ~irst allow the ~ample to traverse the matrix and pass through the binding region, filling up a proportional number of binding sites. One could then add enzyme-antibody conjugate which would bind to antigen captured by antibody in the binding region. The procedure would then follow as described above.
In Fig. 3, i~ depicted an alternate form of a matrix (100). In this matrix, antibodies to red blood cells (102) are bound to the matrLx indicated at ArbC.
Where blood is employed as a sample, the antibodies (102) will ~erve to remove the red blood cell3 ~rom the kraveling sample medium, so that any red blood cell~
will not interfere with the detection of color in the binding region (104). The binding region (104) i~
indicated a~ a circle, but any design may be employed, ~uch a~ a bar, cros3, triangle, or the like. Surround-ing the binding region (104) is control region (106~.
Control region (105) ha3 a predetermined amount of enzyme (110) indicated as E. ~y pefor~ing the as3ay as de~cribed above, the amount of enzyme conjugate bound to the binding region will be related to the amount o~
antigen in the medium. B~ comparing the color produced in the binding region with the color produced in the control region, one can determine whether the a~ount of antigen in the sample exceeds a predetermined level.
This will be particularly useful, where the result i3 either positive or negatiYe~ depending upon whether the - analyte is above a predetermined level.
Figs. 4 and 5 depict a device which may be used for determining a panel of drug3 ~o that a single ~ample may be treated in a variety of ways to give a ~3Q~ 3~3 variety of result~. Panel device (120) is a filter top plate (122) under which appears a plurality of matrixes (124). The individual matrixes may be of the qame or different length3, as required. The matrixes are joined at a central hub region (126). The hub region contacts each of the filter matrices (124) and feeds the ~ample into each matrix. Inlet (128) connects well (130) to the hub (126). The well (130) allows for a measured amount of sample to be added to the well which will then be absorbed into the hub region (126) and be tran~mitted evenl~y to the various filter matrices (124). A plurality o~ reagent addition ports (132) are provided which intimately contact the ends of each matrix (124). Ports (132) are surrounded by a well (134) which allows for the introduction of various reagents through the port. The matrix i9 retained in plate (136), where plate (136) has been cut out to house matrices (124) and hub (126) and further act as a divider between the matrices (124). The plate (136) i9 ~upported by 3eparator plate (138) which intimately contact~ a unidirectional ~low membrane (139), which in turn intimately contacts absorbant material (140). The absorbed fluid components are retained in housing : (144).
In Fig. 6, multiple unit ~150) has a plurality of devices (152) joined together at broken line (154) indicating the presence of a fracture line, allowlng for separation of the individual unit~ de~ired. As in the previous unit~, there is a sample well (156) in the port (158) and a reagent port (160) with well (162) where the ~ilter matrix (164) is indicated by the broken lines.
A~ i~ evident from the above description, the ~ubject device~ and protocols provide for a large num-ber of advantages. As contrasted to other deviceswhich are commercially available, in accordance with the subject invention the sample may be pre-treated ~L3~i~2910 without the necessity for physical removal of a pre-treatment filter, thus avoiding aerosolization of in-fectious or potentially infectious samples. Filtration of sample parallel to the thin dimension of the filter matrix as opposed to perpendicular to the filter matrix as is currently being done or has been depicted in the literature allows for the physical separation of blood cells usins selected materials, such as glass fLber filter~ or filter matrix containing binding ligands, which inhibit the migration of the red blood cells.
The sample may be filtered through a much longer linear dimension of filter material prior to arriving at the binding regionq The ~ilter matrix can be utilized to allow administration, mixing and reaction of immuno-logical components or other pre-treatment components without physical manipulation of these materials.
Thus, user steps may be eliminated from the assay pro-tocol for greater convenience and test reliability. In the subject devices, the matrlx serves as a u~e~ul medium for 3tabilizing dried immunological and pre-treatment reagent~ in the assay device. Since such dried materLal3 are typically more stable than materi-als provided in liquid form, the device~ are more amenable to long ~helf life or 3torage at elevated temperatures. This is a particularly lmportant feature for an over-the-counter or con~umer-oriented product or ior general rield u~e where refrigeration is imprac-tical. In addition, the nature of the ~ilter may be varied in order to allow modulation of the migration time across the matrix to the binding region. This provides a means ior controlling the time of the immu-nological raactions or pre-treatment reactions which czn be provided for with the iilter matrix.
By providing for a long path ~low of fluids, one can allow the sample to traver~e a filter matrix over a relatively lon~ path, or providing for a short path to the binding region for reagents and wash solu-~3~42t3~
2~
tions, where the reag~nts may be provided at a substan-tially constant concentration to the binding region.
The long path of the sample provides for removal of in-terfering materials in an efficient manner. The short path for the wash solutions and reagent provides econo-mies in time, improved control of the contact between reagents, such as conjugates and developer solutions, and may reduce the amount of solution required for ob-taining the desired result.
Multiple test devices allow for performing a panel of related~tests on a single specimen or a series ~batch) of the same tests on a group of different sample~. Multiple test devices provide cost economics in decreasing the cost per reportable result.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of under~tanding, it will be obvious that certain changes and modifications may be practiced within the scope o~ the appended claims-~5 .
3o
Claims (11)
1. A device for the detection of an analyte comprising:
a matrix in a first plane providing for capillary transport of an aqueous sample medium:
cover means positioned over said matrix, a first port in said cover means, with said first port positioned over said matrix for directing said sample medium to a first site on said matrix;
flow means in contact with said matrix at a second site on said matrix for directing flow of said sample medium at said second site to a second plane substantially parallel to said matrix in said first plane, said flow through said flow means being substantially perpendicular to said matrix;
an immunoglobulin bound to said second site; and absorbant means for receiving said sample medium in said second plane.
a matrix in a first plane providing for capillary transport of an aqueous sample medium:
cover means positioned over said matrix, a first port in said cover means, with said first port positioned over said matrix for directing said sample medium to a first site on said matrix;
flow means in contact with said matrix at a second site on said matrix for directing flow of said sample medium at said second site to a second plane substantially parallel to said matrix in said first plane, said flow through said flow means being substantially perpendicular to said matrix;
an immunoglobulin bound to said second site; and absorbant means for receiving said sample medium in said second plane.
2. A device according to Claim 1, including a second port in said cover situated over said second site for di-recting fluids through said second site and said flow means to said absorbant means.
3. A device according to Claim 2. including directing means on a separator support, said separator support substantially parallel to and under said matrix, said directing means in contact with said matrix for linearly directing sample medium from said first site to said second site.
4. A device according to Claim 1, including at least one of:
(a) a second port in said cover means, said second port situated over said second site for directing fluids through said second site to said absorbant means;
(b) directing means in contact with said matrix for linearly directing said sample medium from said first site to said second site; and (c) spring means for urging said matrix, said flow means and said absorbant means toward each other in order to maintain intimate contact.
(a) a second port in said cover means, said second port situated over said second site for directing fluids through said second site to said absorbant means;
(b) directing means in contact with said matrix for linearly directing said sample medium from said first site to said second site; and (c) spring means for urging said matrix, said flow means and said absorbant means toward each other in order to maintain intimate contact.
5. A device for the detection of at least one analyte using a single sample medium, said device comprising:
a plurality of matrixes in a first plane meeting at a central hub, each of said matrixes providing for capillary transport of said sample medium:
cover means positioned over said plurality of matrixes, a first port in said cover means, with said first port positioned over said plurality of matrixes for directing said sample medium to said hub of said plurality of matrixes;
multiple flow means in contact with each of said matrixes at a site on each of said matrixes other than said hub for directing flow of said sample medium at said site on each of said matrixes to a second plane substantially parallel to said plurality of matrixes in said first plane, said flow through said flow means being substantially perpendicular to said plurality of matrixes;
an immunoglobulin bound to each of said matrixes at each of said sites, and absorbant means for receiving said sample medium in said second plane.
a plurality of matrixes in a first plane meeting at a central hub, each of said matrixes providing for capillary transport of said sample medium:
cover means positioned over said plurality of matrixes, a first port in said cover means, with said first port positioned over said plurality of matrixes for directing said sample medium to said hub of said plurality of matrixes;
multiple flow means in contact with each of said matrixes at a site on each of said matrixes other than said hub for directing flow of said sample medium at said site on each of said matrixes to a second plane substantially parallel to said plurality of matrixes in said first plane, said flow through said flow means being substantially perpendicular to said plurality of matrixes;
an immunoglobulin bound to each of said matrixes at each of said sites, and absorbant means for receiving said sample medium in said second plane.
6. A multiunit device for the detection of at least one analyte comprising a plurality of containers, each container sharing at least one common wall with an adjacent container, each container comprising:
a matrix in a first plane providing for capillary transport of an aqueous sample medium:
cover means positioned over said matrix, a first port in said cover means, with said first port positioned over said matrix for directing said sample medium to a first site on said matrix;
flow means in contact with said matrix at a second site on said matrix for directing flow of said sample medium at said second site to a second plane substantially parallel to said matrix in said first plane, said flow through said flow means being substantially perpendicular to said matrix;
an immunoglobulin bound to said matrix at said site; and absorbant means for receiving said sample medium in said second plane.
a matrix in a first plane providing for capillary transport of an aqueous sample medium:
cover means positioned over said matrix, a first port in said cover means, with said first port positioned over said matrix for directing said sample medium to a first site on said matrix;
flow means in contact with said matrix at a second site on said matrix for directing flow of said sample medium at said second site to a second plane substantially parallel to said matrix in said first plane, said flow through said flow means being substantially perpendicular to said matrix;
an immunoglobulin bound to said matrix at said site; and absorbant means for receiving said sample medium in said second plane.
7. A device for the detection of an analyte comprising:
a container including a cover;
a bibulous matrix in a first plane providing for capillary transport of an aqueous sample medium, said bibulous matrix in said container and under said cover;
a first port in said cover, said first port positioned over said bibulous matrix for directing said sample medium to a first site on said matrix;
flow means in contact with said matrix at a second site on said matrix for directing flow of said sample medium at said second site to a second plane substantially parallel to said bibulous matrix in said first plane, said flow through said flow means being substantially perpendicular to said bibulous matrix;
a specific binding pair member bound to said bibulous matrix at said second site; and absorbant means in contact with said flow means extending from said bibulous matrix for receiving said sample medium.
a container including a cover;
a bibulous matrix in a first plane providing for capillary transport of an aqueous sample medium, said bibulous matrix in said container and under said cover;
a first port in said cover, said first port positioned over said bibulous matrix for directing said sample medium to a first site on said matrix;
flow means in contact with said matrix at a second site on said matrix for directing flow of said sample medium at said second site to a second plane substantially parallel to said bibulous matrix in said first plane, said flow through said flow means being substantially perpendicular to said bibulous matrix;
a specific binding pair member bound to said bibulous matrix at said second site; and absorbant means in contact with said flow means extending from said bibulous matrix for receiving said sample medium.
8. A device according to Claim 7, wherein said specific binding pair member is an immunoglobulin.
9. A method for determining an analyte in a sample medium, said method comprising:
contacting said sample medium at a first site on a matrix in a first plane, said matrix capable of transporting said sample medium by capillary transport;
directing said sample medium through said matrix toward a second site on said matrix distant from said first site, wherein a specific binding pair member is bound at said second site;
directing said sample medium through said second site to a second plane substantially parallel to said first plane, where a complex is formed between said specific binding pair member and its homologous binding pair member; and determining the presence of complex formation at said second site as indicating presence of said analyte in said sample.
contacting said sample medium at a first site on a matrix in a first plane, said matrix capable of transporting said sample medium by capillary transport;
directing said sample medium through said matrix toward a second site on said matrix distant from said first site, wherein a specific binding pair member is bound at said second site;
directing said sample medium through said second site to a second plane substantially parallel to said first plane, where a complex is formed between said specific binding pair member and its homologous binding pair member; and determining the presence of complex formation at said second site as indicating presence of said analyte in said sample.
10. A method according to Claim 9, wherein said determining comprises passing a solution of an enzyme conjugate capable of binding directly or indirectly with said analyte through said second site, followed by passing a solution of substrate for said enzyme through said second site, whereby said enzyme converts said substrate to a product providing a visual signal.
11. A method according to Claim 9, wherein said second site has anti-enzyme bound to said matrix, and between said first and second sites is a first zone of anti-analyte or analyte analog bound to said matrix, and including the additional step of adding analyte analog-enzyme conjugate to said sample medium before said sample medium contacts said first zone.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US006,662 | 1987-01-23 | ||
| US07/006,662 US5079142A (en) | 1987-01-23 | 1987-01-23 | Orthogonal flow immunoassays and devices |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1304290C true CA1304290C (en) | 1992-06-30 |
Family
ID=21721997
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000557095A Expired - Fee Related CA1304290C (en) | 1987-01-23 | 1988-01-21 | Orthographic flow immunoassays and devices |
Country Status (3)
| Country | Link |
|---|---|
| US (1) | US5079142A (en) |
| EP (1) | EP0276152A3 (en) |
| CA (1) | CA1304290C (en) |
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| US10634672B2 (en) | 2014-10-07 | 2020-04-28 | 3M Innovative Properties Company | Method of detecting a microorganism using chromatographic enrichment |
| US11061026B2 (en) | 2017-02-17 | 2021-07-13 | MFB Fertility, Inc. | System of evaluating corpus luteum function by recurrently evaluating progesterone non-serum bodily fluids on multiple days |
| WO2019152657A1 (en) | 2018-02-03 | 2019-08-08 | Simple Healthkit, Inc. | Reliable, comprehensive, and rapid sexual health assessment |
Family Cites Families (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA983358A (en) * | 1971-09-08 | 1976-02-10 | Kenneth D. Bagshawe | Performance of chemical or biological reactions |
| US4235601A (en) * | 1979-01-12 | 1980-11-25 | Thyroid Diagnostics, Inc. | Test device and method for its use |
| US4361537A (en) * | 1979-01-12 | 1982-11-30 | Thyroid Diagnostics, Inc. | Test device and method for its use |
| US4323536A (en) * | 1980-02-06 | 1982-04-06 | Eastman Kodak Company | Multi-analyte test device |
| US4366241A (en) * | 1980-08-07 | 1982-12-28 | Syva Company | Concentrating zone method in heterogeneous immunoassays |
| US4938927A (en) * | 1982-01-08 | 1990-07-03 | Environmental Diagnostics, Inc. | Rotary fluid manipulator |
| US4435504A (en) * | 1982-07-15 | 1984-03-06 | Syva Company | Immunochromatographic assay with support having bound "MIP" and second enzyme |
| US4632901A (en) * | 1984-05-11 | 1986-12-30 | Hybritech Incorporated | Method and apparatus for immunoassays |
| DE3445816C1 (en) * | 1984-12-15 | 1986-06-12 | Behringwerke Ag, 3550 Marburg | Flat diagnostic agent |
| US4753775A (en) * | 1985-04-12 | 1988-06-28 | E. I. Du Pont De Nemours And Company | Rapid assay processor |
| US4803170A (en) * | 1985-05-09 | 1989-02-07 | Ultra Diagnostics Corporation | Competitive immunoassay method, device and test kit |
| IL78854A (en) * | 1985-05-31 | 1991-11-21 | Murex Corp | Biological fluid diagnostic device |
| US4670381A (en) * | 1985-07-19 | 1987-06-02 | Eastman Kodak Company | Heterogeneous immunoassay utilizing horizontal separation in an analytical element |
| US4756844A (en) * | 1986-12-29 | 1988-07-12 | The Dow Chemical Company | Controlled-release composition having a membrane comprising submicron particles |
| CA1313616C (en) * | 1987-06-01 | 1993-02-16 | Robert B. Sargeant | Lateral flow, non-bibulous membrane protocols |
| US4943522A (en) * | 1987-06-01 | 1990-07-24 | Quidel | Lateral flow, non-bibulous membrane assay protocols |
| US4981786A (en) * | 1987-09-04 | 1991-01-01 | Syntex (U.S.A.) Inc. | Multiple port assay device |
| US5006474A (en) * | 1987-12-16 | 1991-04-09 | Disease Detection International Inc. | Bi-directional lateral chromatographic test device |
-
1987
- 1987-01-23 US US07/006,662 patent/US5079142A/en not_active Expired - Fee Related
-
1988
- 1988-01-21 CA CA000557095A patent/CA1304290C/en not_active Expired - Fee Related
- 1988-01-21 EP EP88300476A patent/EP0276152A3/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| US5079142A (en) | 1992-01-07 |
| EP0276152A3 (en) | 1990-06-27 |
| EP0276152A2 (en) | 1988-07-27 |
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