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Publication numberUS20030224366 A1
Publication typeApplication
Application numberUS 10/147,679
Publication dateDec 4, 2003
Filing dateMay 16, 2002
Priority dateNov 17, 1999
Also published asCA2392009A1, CA2392009C, DE60025529D1, DE60025529T2, EP1232502A1, EP1232502B1, US8129118, US20050266462, US20120247150, WO2001037291A1
Publication number10147679, 147679, US 2003/0224366 A1, US 2003/224366 A1, US 20030224366 A1, US 20030224366A1, US 2003224366 A1, US 2003224366A1, US-A1-20030224366, US-A1-2003224366, US2003/0224366A1, US2003/224366A1, US20030224366 A1, US20030224366A1, US2003224366 A1, US2003224366A1
InventorsKurt Weindel, Michael Riedling, Albert Geiger
Original AssigneeKurt Weindel, Michael Riedling, Albert Geiger
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Magnetic glass particles, method for their preparation and uses thereof
US 20030224366 A1
Abstract
This invention relates to magnetic particles having a glass surface which are substantially spherical. This invention also relates to methods for making them, as well as to suspensions thereof and their uses for the purification of DNA or RNA in particular in automated processes.
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Claims(64)
1. A composition of magnetic glass particles
wherein
the magnetic glass particles comprise at least one magnetic object with a mean diameter between 5 to 500 nm.
2. The composition of claim 1
wherein
the magnetic object has a mean diameter between 10 to 200 nm, preferably between 15 to 50 nm.
3. The composition of claim 1
wherein
the half-life period for the sedimentation of a 3 mg/ml weight-per-volume suspension of the composition in isopropanol is more than 3 min.
4. The composition of claim 1
wherein
the half-life period is more than 6 min.
5. The composition of claim 1
wherein
the magnetic object has a mean diameter of 23 nm.
6. The composition of claim 1
wherein
the diameter ratio of the magnetic body to the glass shell is less than 1 to 10.
7. The composition of claim 1
wherein
the magnetic glass particles have a mean diameter between 0.5 μm and 5 μm.
8. The composition of claim 7
wherein
the magnetic glass particles have a mean diameter between 1 μm to 2 μm.
9. The composition of claim 1
wherein
the magnetic glass particles are microporous.
10. The composition of claim 1
wherein
the pore surface of the magnetic glass particles is less than 10% of the total surface.
11. The composition of claim 1
wherein
the magnetic object is superparamagnetic.
12. The composition of claim 1
wherein
the magnetic object is ferrimagnetic or ferromagnetic.
13. The composition of claim 1
wherein
the magnetic object comprises iron or iron oxide.
14. The composition of claim 13
wherein
the iron oxide is Fe3O4 or γ-Fe2O3.
15. The composition of claim 1
wherein
the magnetic glass particles have a surface area of more than 4 m2/g.
16. The composition of claim 15
wherein
the magnetic glass particles have a surface area between 5 to 100 m2/g, preferably in the range of 10 to 50 m2/g, most preferably in the range of 15 to 30 m2/g.
17. The composition of claim 1
wherein
the magnetic glass particles are substantially spherical.
18. A suspension comprising a composition of magnetic glass particles according to claim 1 in a liquid.
19. The suspension of claim 18
wherein
the liquid comprises an alcohol.
20. The composition of claim 19
wherein
the alcohol is isopropanol or ethanol.
21. The composition of claim 19
wherein
the liquid is 70% (V/V) ethanol.
22. The suspension of claim 18
wherein
the liquid is a buffered aqueous solution.
23. The suspension of claim 22
wherein
the solution is buffered by Tris-hydroxymethylamine, phosphate, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) or a salt thereof.
24. The suspension of claim 22
wherein
the solution further contains substances modifying the ionic strength or metal-complexing agents.
25. The suspension of claim 22
wherein
the suspension additionally contains DNA or RNA.
26. The suspension of claim 22
wherein
the suspension contains additionally DNA or RNA or both in a mixture with one or more compounds selected from the group of proteins, fatty acids, carbohydrates or any other biological material.
27. The suspension of claim 18
wherein
the suspension additionally contains a chaotropic agent.
28. The suspension of claim 27
wherein
the chaotropic agent is selected from the group consisting of sodium iodite, sodium perchlorate, guanidinium thiocyanate, guanidinium isothiocyanate or guanidinium hydrochloride.
29. The suspension of claim 27
wherein
the chaotropic agent is present in a concentration between 2 and 8 mol/l, and preferably between 4 and 6 mol/l.
30. The suspension of claim 18
wherein
the suspension contains 5 to 60 mg/ml of a composition of magnetic glass particles
wherein
the magnetic glass particles comprise at least one magnetic object with a mean diameter between 5 to 500 nm.
31. A tube containing the composition of claim 1.
32. A storage container containing the composition of claim 1.
33. A kit of parts comprising the tube of claim 1 or the storage container of claim 1.
34. The kit of parts of claim 1 further comprising a washing solution or an eluent.
35. The kit of parts of claim 1 further comprising reagents suitable for the purification of a nucleic acid.
36. A tube containing the suspension of claim 18.
37. A storage container containing the suspension of claim 18.
38. A kit of parts comprising the tube of claim 36 or the storage container of claim 37.
39. A method for the production of a composition of magnetic glass particles comprising the steps of
a) suspending magnetic objects with a diameter between 5 and 500 nm in a sol,
b) spray-drying the suspension in a two-nozzle spray-drier, and
c) sintering the spray-dried powder.
40. The method of claim 39
wherein
the inlet temperature of the two-nozzle spray-drier is between 120° C. and 500° C.,
the outlet temperature is chosen according to the boiling point of the sol and the spray pressure is at least 3 bar.
41. The method of claim 39
wherein
the inlet temperature of the two-nozzle spray-drier is between 170° C. and 230° C., preferably between 190° C. and 210° C.
42. The method of claim 39
wherein
the inlet temperature of the two-nozzle spray-drier is 200° C.
43. The method of claim 39
wherein
the spray pressure is between 4 and 6 bar.
44. The method of claim 39
wherein
the sol contains ethanol as solvent.
45. The method of claim 44
wherein
the outlet temperature is between 50° C. and 300° C.
46. The method of claim 44
wherein
the outlet temperature is between 90° C. and 100° C.
47. The method of claim 39
wherein
the sinter temperature is between 400° C. to 1200° C., preferably between 720° C. and 770° C.
48. The magnetic glass particles produced by the method of claim 1.
49. A procedure for isolating a biological material comprising
a) contacting a sample that contains the biological material in a liquid with a composition of magnetic glass particles wherein the magnetic glass particles comprise at least one magnetic object with a mean diameter between 5 to 500 nm under conditions in which the biological material binds directly to the surface of the magnetic glass particles, and
b) separating the biological material from the liquid.
50. The procedure of claim 49 wherein the biological material is a nucleic acid.
51. The procedure of claim 49 wherein the biological material contains a mixture of nucleic acids, wherein the target nucleic acid or target nucleic acids is/are present in low abundance.
52. The procedure of claim 49 wherein
the separation is accomplished with the aid of a magnet.
53. The procedure of claim 49 wherein
the magnetic particles are not premagnetized when brought into contact with the sample.
54. The procedure of claim 49 wherein the procedure is automatized.
55. The procedure of claim 49 wherein
the procedure is in a high-throughput format.
56. The procedure of claim 49 wherein
during the procedure a suspension of magnetic glass particles, wherein the magnetic glass particles comprise at least one magnetic object with a mean diameter between 5 to 500 nm, is taken from a storage container and partial volumes of the suspension are added to different reaction vessels.
57. The procedure of claim 49 wherein
the nucleic acid is detected after the purification.
58. The procedure of claim 49 wherein the nucleic acid is detected after an amplification step.
59. The procedure of claim 57
wherein
the nucleic acid is detected by a process comprising:
a) contacting the sample with an oligonucleotide comprising a sequence complementary to a region of the target nucleic acid and a labeled oligonucleotide comprising a sequence complementary to a second region of the same target nucleic acid sequence strand, but not including the nucleic acid sequence defined by the first oligonucleotide, to create a mixture of duplexes during hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the first oligonucleotide and to the labeled oligonucleotide such that the 3′-end of the first oligonucleotide is adjacent to the 5′-end of the labeled oligonucleotide;
b) maintaining the mixture of step a) with a template-dependent nucleic acid polymerase having a 5′ to 3′ nuclease activity under conditions sufficient to permit the 5′ to 3′ nuclease activity of the polymerase to cleave the annealed, labeled oligonucleotide and release labeled fragments; and
c) detecting and/or measuring the signal generated by the hydrolysis of the labeled oligonucleotide.
60. The procedure of claim 59
wherein
the detection is carried out in the presence of a blocking oligonucleotide.
61. The procedure of claim 60
wherein
the blocking oligonucleotide is an aptamer.
62. The procedure of claim 61
wherein
the aptamer has the sequence SEQ ID NO: 23.
63. The procedure of claim 60
wherein the amplification and detection reaction is carried out in a homogeneous solution-phase multiplex assay format for the simultaneous detection of multiple targets.
64. The procedure of claim 60
wherein for at least 5 cycles of the polymerase chain reaction, the annealing temperature is less than 8° C., preferably less than 3° C. above the dissociation temperature of the polymerase-aptamer complex.
Description
  • [0001]
    This invention relates to magnetic particles having a glass surface and are substantially spherical. This invention also relates to methods for making them, as well as to suspensions thereof and their uses for the purification of biological material in particular in automated processes.
  • [0002]
    Many biological materials, especially nucleic acids, present special challenges in terms of isolating them from their natural environment. On the one hand, they are often present in very small concentrations and, on the other hand, they are often found in the presence of many other solid and dissolved substances that make them difficult to isolate or to measure, in particular in biospecific assays.
  • [0003]
    Biospecific binding assays allow of the detection of specific analytes, e.g. nucleic acids, or specific analyte properties and play a major role in the field of diagnostics and bioanalytics. Examples therefor are hybridisation assays, immuno assays and receptor-ligand assays.
  • [0004]
    Hybridisation assays make use of the specific base-pairing for the molecular detection of nucleic acid analytes e.g. RNA and DNA. Hence, oligonucleotide probes with a length of 18 to 20 nucleotides may enable the specific recognition of a selected sequence in the human genome. Another assay which makes use of the selective binding of two oligonucleotide primers is the polymerase chain reaction (PCR) described in U.S. Pat. No. 4,683,195. This method makes use of the selective amplification of a specific nucleic acid region to detectable levels by a thermostable polymerase in the presence of desoxynucleotide triphosphates in several cycles.
  • [0005]
    Nucleic acids are comparatively complex analytes which have normally to be extracted from complex mixtures before they can be used in a probe-based assay.
  • [0006]
    There are several methods for the extraction of nucleic acids:
  • [0007]
    sequence-dependent or biospecific methods as e.g.:
  • [0008]
    affinity chromatography
  • [0009]
    hybridisation to immobilised probes on beads
  • [0010]
    sequence-independent or physico-chemical methods as e.g.:
  • [0011]
    liquid-liquid extraction with e.g. phenol-chloroform
  • [0012]
    precipitation with e.g. pure ethanol
  • [0013]
    extraction with filter paper
  • [0014]
    extraction with micelle-forming agents as cetyl-trimethyl-ammonium-bromide
  • [0015]
    binding to immobilised, intercalating dyes, e.g. acridine derivatives
  • [0016]
    adsorption to silica gel or diatomic earths
  • [0017]
    adsorption to magnetic glass particles (MGP) or organo silane particles under chaotropic conditions
  • [0018]
    Many procedures and materials for isolating nucleic acids from their natural environment have been proposed in recent years by the use of their binding behavior to glass surfaces. In Proc. Natl. Acad. USA 76, 615-691 (1979), for instance, a procedure for binding nucleic acids in agarose gels in the presence of sodium iodide in ground flint glass is proposed.
  • [0019]
    The purification of plasmid DNA from bacteria on glass dust in the presence of sodium perchlorate is described in Anal. Biochem. 121, 382-387 (1982).
  • [0020]
    In DE-A 37 34 442, the isolation of single-stranded M13 phage DNA on glass fiber filters by precipitating phage particles using acetic acid and lysis of the phage particles with perchlorate is described. The nucleic acids bound to the glass fiber filters are washed and then eluted with a menthol-containing buffer in Tris/EDTA buffer.
  • [0021]
    A similar procedure for purifying DNA from lambda phages is described in Anal. Biochem. 175, 196-201 (1988).
  • [0022]
    The procedure known from the prior art entails the selective binding of nucleic acids to glass surfaces in chaotropic salt solutions and separating the nucleic acids from contaminants such as agarose, proteins or cell residue. To separate the glass particles from the contaminants according to the prior art, the particles are either centrifuged or fluids are drawn through glass fiber filters. This is a limiting step, however, that prevents the procedure from being used to process large quantities of samples.
  • [0023]
    It has been demonstrated that magnetic particles covered with a glass surface offer considerable advantages for isolating biological materials. If the magnetic particles have not been brought in contact with a magnetic field, gravity is the only force that can cause them to sediment out. They can be resuspended by shaking the solution. The sedimentation procedure that does not utilize a magnetic field proceeds more slowly than the immobilization of biological materials on the surface of the particles. This is especially true for nucleic acids. The magnetic particles can be easily collected at a specific location in the sample fluid by means of a magnet. The fluid is then separated from the particles and, therefore, from the immobilized biological materials.
  • [0024]
    The use of magnetic particles to immobilize nucleic acids after precipitation by adding salt and ethanol is described in Anal. Biochem. 201, 166-169 (1992) and PCT GB 91/00212. In this procedure, the nucleic acids are agglutinated along with the magnetic particles. The agglutinate is separated from the original solvent by applying a magnetic field and performing a wash step. After one wash step, the nucleic acids are dissolved in a Tris buffer. This procedure has a disadvantage, however, in that the precipitation is not selective for nucleic acids. Rather, a variety of solid and dissolved substances are agglutinated as well. As a result, this procedure can not be used to remove significant quantities of any inhibitors of specific enzymatic reactions that may be present.
  • [0025]
    Magnetic, porous glass is also available on the market that contains magnetic particles in a porous, particular glass matrix and is covered with a layer containing streptavidin. This product can be used to isolate biological materials, e.g., proteins or nucleic acids, if they are modified in a complex preparation step so that they bind covalently to biotin. Magnetizable particular adsorbents proved to be very efficient and suitable for automatic sample preparation. Ferrimagnetic and ferromagnetic as well as superparamagnetic pigments may be used for this purpose.
  • [0026]
    Particles, according to the expert, are solid materials having a small diameter. Particles like these are often also referred to as pigments.
  • [0027]
    Those materials are referred to as magnetic that are drawn to a magnet, i.e., ferromagnetic or superparamagnetic materials, for instance. Superparamagnetism is seen as advantageous and preferable in the state of the art (e.g. U.S. Pat. No. 5,928,958; U.S. Pat. No. 5,925,573; EP 757 106). The glass or organosilane surfaces are often functionalised in order to be used for biospecific capture reactions, e.g. U.S. Pat. No. 5,928,958, U.S. Pat. No. 5,898,071, U.S. Pat. No. 5,925,573, EP 937 497, U.S. Pat. No. 4,554,088 or U.S. Pat. No. 4,910,148. Alternatively, glass or organosilane surfaces may be treated with various solvents or salts to modify their hydrophilicity and/or electropositivity, e.g. U.S. Pat. No. 5,438,127.
  • [0028]
    However, the underivatized silanol groups of the glass or the silane surface may be used for the adsorption with pure physico-chemical forces under suitable reaction conditions as described in DE 195 20 964, DE 195 37 985, WO 96/41840, WO 96/41811, EP 757 106 or U.S. Pat. No. 5,520,899. Typically, magnetic cores or magnetic core aggregates are covered with a glass surface which is formed by an acid- or base-catalyzed sol-gel-process. These particles are called core-shell particles. The glass shell then has a typical layer thickness (see e.g. DE 195 20 964) wherein the size and shape of the pigment, which may contain a non-magnetic support as e.g. mica in addition to the magnetic metal oxide, determines size and form of the produced particle (see e.g. DE 195 37 985 and corresponding WO 96/41811). To obtain a high surface activity, glass material with a high porosity is used (see e.g. EP 757 106; WO 99/26605). Further, composite magnetic particles are described, e.g. silicate-covered ferric oxide covered with an inorganic silica matrix from silica particles (EP 757 106) or mixtures of glass and silica gel (WO95/06652).
  • [0029]
    The problem to be solved by the present invention can be seen as providing magnetic glass particles with improved properties for sample preparation and for biological assays, in particular for automated processes.
  • [0030]
    The deficiencies of the magnetic glass particles in the state of the art are overcome by the findings of the present invention.
  • [0031]
    It is an object of the invention to provide a composition of magnetic glass particles. The magnetic glass particles (MGPs) according to the present invention are a solid dispersion of small magnetic cores in glass. The MGPs are comparatively small and are substantially spherical. The non-magnetic fine content of a composition of the MPGs is very low because of the method of their preparation. This has the effect that suspensions of the MGPs sediment slowly and can therefore be advantageously used for processes in molecular biology which can be automatized. In one embodiment of the invention compositions and suspensions of the MGPs according to the present invention are provided. In another embodiment of the invention a method for the composition of the MGPs is provided. In still another embodiment of the invention a method for the purification of DNA or RNA is provided in which the MGPs according to the present invention are used.
  • [0032]
    It is a object of the present invention to provide a composition of magnetic glass particles which are substantially spherical and have a small diameter and contain at least one magnetic object with a diameter between 5 and 500 nm. This has surprising consequences on the sedimentation kinetics, quantified by the half time values t1/2, which is the time span until 50% of the particles have sedimented from a specific volume element (see Example 6). The half-life period for the sedimentation of a 3 mg/ml weight-per-volume suspension of the composition in isopropanol is more than 3 min, preferably 4 min, more preferably 6 min. However the most preferred values for the half-life period is more than 10 min or even more than 20 min. The smaller and closer to the ideal sphere, the longer the MGPs will be suspended. This may be explained by the fact that the closer the form will resemble an ideal sphere, the lower the possibility that two or more particles will stick together and built up aggregates which may sediment more rapidly. These data are shown in Example 6 and high resolution scanning electron microscopical images can be seen in FIG. 4 to FIG. 10. This has the advantage that for automated processes the required mixing intensity and mixing frequency of the storage containers containing the MGP suspension is reduced as the repetitive dosage of a specific MGP suspension volume from a surplus volume sucked into a syringe is easier (more precise delivery with regard to massMGP/volume).
  • [0033]
    The MGPs according to the present invention are glass droplets in which very small non-aggregating magnetic objects are dispersed. Those objects that are referred to as magnetic are drawn to a magnet, i.e., ferromagnetic or superparamagnetic materials, for instance. Preferred are ferromagnetic materials, in particular if they have not yet been premagnetized. Premagnetization in this context is understood to mean bringing in contact with a magnet, which increases the remanence. Preferred magnetic materials are iron or iron oxide as e.g. magnetite (Fe3O4) or Fe2O3, preferably γ-Fe2O3. In principle, barium ferrite, nickel, cobalt, Al—Ni—Fe—Co alloys or other feri- or ferromagnetic could be used. The magnetic objects may be e.g. a magnetic pigment. The size of the magnetic objects is in the nanoscale range, i.e. according to the present invention the diameter is between 5 to 500 nm, preferably between 10 to 200 nm, most preferably between 15 to 50 nm. Suitable magnetic pigments are manufactured by the company CERAC which have a mean diameter of 23 nm and consist of γ-Fe2O3 (BET-surface 50 m2/g, CERAC: P.O. Box 1178, Milwaukee, Wis. 53201-1178 USA; Article-No. I-2012). The magnetic glass particles according to the present invention are further characterized by the fact that the MGPs have a particle diameter between 0.5 μm and 5 μm, preferably between 1 μm to 2 μm as determined by high resolution scanning electron microscopy, whereas the magnetic objects have a diameter between 5 to 500 nm, preferably between 10 to 200 nm, most preferably in the range of 15 to 50 nm as said above. Hence, the MGPs of the present invention are further characterized by a diameter ratio of magnetic pigment core to magnetic glass particle of less than 1 to 10 as determined by high resolution scanning electron microscopy. Because of these diameter ratios as well as the absence of any inert carrier that would determine shape and size of the particles, the geometry of the MGPs and the number of incorporated magnetic objects, are determined by the conditions of manufacturing. The MGPs according to the present invention are microporous but have highly-structured and therefore relatively large surface with more than 6 m2/g. Preferably, the magnetic glass particles according to the present invention have a surface area in the range of 5 to 100 m2/g, preferably 5 to 90 m2/g, more preferably in the range of 10 to 50 m2/g, most preferably in the range of 15 to 30 m2/g. This surface is approximately double the size of the particles described in DE 195 37 985. This can be determined by the Braunauer-Emett-Teller-method using an automated commercial apparatus (see Example 4). For a discussion of this method, familiarly called the BET method, see S. Braunauer. The Adsorption of Gases and Vapors, Vol. 1, Princeton University Press, 1943. For example, the sample EJ0096.5R-01 which is of preferential interest (see Example 1 and Table 1 to Table 3 for a summary of the production parameters) has a BET-surface of 26.8525 m2/g, a micropore area of 2.3058 m2/g and an average pore diameter of 24.9132 nm. This means that the pore surface is less than 10% of the total surface and that the magnetic glass particle is microporous.
  • [0034]
    A pore is understood to be a recess in the outer surface of the particle. The surface reaches so far into the particle that a perpendicular line drawn in the recess on the surface cuts the particle at least once in the direction of the adjacent environment of the particle. In addition, pores reach into the particle to a depth that is greater than one radius of the pore.
  • [0035]
    The slower sedimentation kinetics, larger surface and the aggregation-inhibiting spherical form manifest themselves in the better functional performance as adsorbent in the nucleic acid diagnosis (see Example 3, 5 and 7) when compared to the German patent applications DE 198 54 973.3 or DE 198 55 259.9. This criterion can be quantified by a shift of the threshold cycles in so-called TaqMan® assays, the signal-to-noise ratio and of the statistically validated lower detection limit. The methods for this assay are disclosed in WO92/02638 and the corresponding US patents U.S. Pat. No. 5,210,015, U.S. Pat. No. 5,804,375, U.S. Pat. No. 5,487,972). Radiotracing experiments (see Example 5.2) showed that the binding behavior with regard to DNA and RNA was the same when compared to reference material known in the state of the art. Surprisingly, the production parameters had an influence on the performance in the radiotracing experiments. A further advantage of the MGP-type of the present invention is that no tensions in the glass layer can lead to fissure during the drying process and corresponding damages in the glass shell because of the inner structure (solid dispersion of small magnetic cores in a glass drop). This can be investigated by image-producing methods (see Example 3).
  • [0036]
    Another embodiment of the present invention is a suspension of magnetic particles. It is obvious for the person skilled in the art to produce a suspension by adding a liquid to a composition of the MGPs and mix the suspension to homogeneity. A liquid according to the present invention may comprise any liquid which does not affect the stability of the magnetic particles and may be used to produce a homogenous suspension. Preferably liquids are used which are suitable for processes in molecular biology, in particular desoxyribonucleic acid (DNA) or ribonucleic acid (RNA) purification processes which make use of the binding of these substances to glass particles under certain conditions. Preferred liquids comprise alcohols or any mixtures thereof with water or ketones. Alcohols shall include according to the invention preferably primary, secondary or tertiary alcohols of the general formula R—OH where the R stands for the general formula —(—CH2)n—CH3 with n>=0. However, other alcohols can also be used if they are suitable for molecular biology purposes as e.g. glycerol. Particularly suitable are the alcohols isopropanol, ethanol or mixtures thereof with water, preferably a mixture of 80 volume parts of isopropanol with 20 volume parts of water (. In another embodiment of the invention the liquid comprises ketones as e.g. acetone. In a preferred embodiment of the invention these suspensions contain between 5 to 60 mg/ml MGPs. In another embodiment of the invention the MGPs are suspended in aqueous buffered solutions which may optionally contain a chaotropic agent in a concentration of between 2 and 8 mol/l, and preferably between 4 and 6 mol/l. Chaotropic salts can be sodium iodide, sodium perchlorate, guanidinium thiocyanate, guanidinium isothiocyanate or guanidinium hydrochlorite. Other compounds are also possible. A chaotropic agent according to the present invention will be any chemical substance which will disturb the ordered structure of liquid water and will have the effect that DNA or RNA will bind to the MGPs according to the present invention if this agent is present in the DNA or RNA containing solution. It is obvious for the artisan to produce suitable aqueous buffered solutions. Buffer systems which may be used for molecular biology purposes may be found e.g. in Sambrook et al. (1989), Molecular Cloning, Cold Spring Harbor University Press, New York, N.Y., USA. Preferred buffer substances are Tris-hydroxymethylamine (TRIS), phosphate, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), salts thereof or other suitable substances. Additionally, substances may be present which modify the ionic strength of the solution as e.g. NaCl, KCl or CaCl2 or which are metal cation complexing agents as e.g. ethylene-diamine-tetra-acetic acid (EDTA) or the salts thereof. Biological material known to the expert in the field may also be present. In another embodiment of the invention the suspension of MGPs may additionally contain DNA or RNA optionally in a mixture with proteins, fatty acids, carbohydrates and other material from biological origin. In another embodiment of the invention the liquid may contain a mixture of one or more constituents selected from the group of alcohols, ketones, aqueous buffered solutions, chaotropic agents, substances which modify the ionic strength of the solution, complexing agents, biological material, DNA or RNA all with the features as described above.
  • [0037]
    In another embodiment of the invention a tube or reaction vessel containing the suspension according to the invention is provided. The tube can be made of plastics but it may also be part of a larger structure, e.g. part of a microtitreplate in 96- or 384-well-format. In still another embodiment of the invention a storage container is provided which contains a composition of magnetic glass particles or suspensions thereof. In another embodiment of the invention a kit of parts is provided which comprises a storage container containing the magnetic glass particles or a suspension thereof according to the present invention. The kit may be used for the purification of DNA or RNA. Such kits known in the art further comprise plastics ware which may be used during the purification procedure as e.g. microtitreplates in the 96 or 384 well format or just ordinary reaction tubes manufactured e.g. by Eppendorf, Hamburg, Germany. The kit may further comprise a washing solution which is suitable for the washing step of the magnetic glass particles when DNA or RNA is bound thereto. Often the washing solution is provided as a stock solution which has to be diluted before the use. The kit may further comprise an eluent, i.e. a solution or a buffer (e.g. TE, 10 mM Tris, 1 mM EDTA, pH 8.0) or pure water to elute the DNA or RNA bound to the magnetic glass particles. Further, additional reagents may be present which can be used for the purification process of a nucleic acid, i.e. DNA or RNA. In one embodiment of the invention the kit of parts according to the present invention is used for the purification of a nucleic acid.
  • [0038]
    In one embodiment of the invention the composition of MGPs may be used to produce a suspension as already described.
  • [0039]
    In another embodiment of the invention the suspensions according to the present invention may be used for the purification of nucleic acids, i.e. RNA or DNA, from complex mixtures with other biological substances containing them. Thereby also mixtures of different nucleic acids may be purified, even mixtures containing a nucleic acid of interest in low abundance. The purification effect results from the behavior of DNA or RNA to bind to magnetic glass particles under certain conditions e.g. in the presence of certain concentration of a chaotropic agent. Preferably, the MGPs with the bound DNA or RNA are washed afterwards at least once, preferably with a mixture of 70 volume parts ethanol with 30 volume parts water (“70% Ethanol”). Afterwards, the conditions are reversed, e.g. the concentration of the chaotropic agent is decreased, to elute the DNA or RNA bound to the MGP the particle. Preferably, this is done by the pelleting of the magnetic glass particles, e.g. by gravity force or by the use of a magnet, and resuspending in a solution with no or only a low amount of chaotropic agent. Alternatively, the solution can be diluted with a solution with no or only a low amount of chaotropic agent. The purified DNA or RNA can now be used for other reactions.
  • [0040]
    It is one object of the invention to provide a production method for the MGPs according to the present invention. A glass according to the present invention is understood to be an amorphous material that contains silicium. Glass can contain other materials such as B2O3 (0-30%), Al2O3 (0-20%), CaO (0-20%), BaO (0-10%), K2O (0-20%), Na2O (0-20%), MgO (0-18%), Pb2O3 (0-15%). Glass can also contain a smaller percentage (0-5%) of a number of other oxides such as Mn2O3, TiO2, As2O3, Fe2O3, CuO, CoO, etc.
  • [0041]
    Especially preferred according to the present invention are glasses that are formed using the gel sol process described in WO 96/41811 and then dried and compressed. The basic principles of this process are known and were described, for instance, in C. J. Brinker, G. W. Scherer “Sol Gel Science—The Physics and Chemistry of Sol Gel Processing”, Academic Press Inc. 1990, Sol-Gel Optics, Processing and Applications, Lisa C. Klein, Ed., Kluwer Academic Publishers 1994, p. 450 ff., and in DE-A-1941191, DE-A-3719339, DE-A-4117041, DE-A-4217432 and WO96/41811. Principally, in the gel-sol process, alkoxides of network-forming components, e.g., SiO2, B2O3, Al2O3, TiO2, ZrO2, GeO2, are combined with oxides and salts of other components, e.g., in an alcohol solution, and then hydrolized. The equation below describes the procedure for making sodium boroaluminium silicate glass:
  • [0042]
    Water is added to begin the hydrolysis process of the starting components. The reaction proceeds relatively quickly because the alkali ions have a catalytic effect on the speed of hydrolysis of the silicic acid ester. Once the gel is formed it can be dried and densified (or condensed) by means of a thermal process to form glass.
  • [0043]
    In one embodiment of the invention the glass matrix will be produced by acid or base catalyzed sol-gel synthesis as shown schematically in FIG. 1 and FIG. 2 and described in detail in Example 1. Here, use is made of colloidal systems wherein at first the solid constituents are dispersed in the liquid phase (=sol) and after the processing they are interconnected like a honeycomb pattern (=gel). The composition of the glass (code EJ) as calculated from the quantity of the educts was 70.67 Mol % SiO2, 14.33 Mol % B2O3, 5.00 Mol % Al2O3, 4.00 Mol % K2O, 2.00 Mol % CaO, 4.00 Mol % ZnO. The composition of the glass (code RN) was 74 Mol % SiO2, 15 Mol % B2O3, 5.00 Mol % Al2O3, 4.00 Mol % K2O, 2.00 Mol % CaO. The composition of the glass (code EP) was 73.61 Mol % SiO2, 14.93 Mol % B2O3, 5.21 Mol % Al2O3, 4.17 Mol % K2O, 2.08 Mol % CaO.
  • [0044]
    The reaction may be described as follows:
  • [0045]
    either acid-catalyzed, e.g.:
  • [0046]
    BCl3+3H2O→B(OH)3+3H(+)Cl(−)
  • [0047]
    AlCl3+3H2O→Al(OH)3+3H(+)Cl(−)
  • [0048]
    SiCl4+4H2O→Si(OH)4+4H(+)Cl(−)
  • [0049]
    Na(+)NO3 (−)+H2O→Na(+)OH(−)+H(+)NO3 (−)
  • [0050]
    K(+)(−)OOCCH3+H2O→K(+)OH(−)+H(+)(−)OOCCH3
  • [0051]
    or alkaline-catalyzed, e.g.
  • [0052]
    K(+)(−)OCH2CH3+H2O→K(+)OH(−)+HOCH2CH3
  • [0053]
    Na(+)(−)OCH3+H2O→Na(+)OH(−)+HOCH3
  • [0054]
    Al(3+)[(−)OCH2CH2CH3]3+3H2O→Al(OH)3+3HOCH2CH2CH3
  • [0055]
    B(OCH2CH3)3+3H2O→B(OH)3+3HOCH2CH3
  • [0056]
    The diverse hydroxides condensate to the corresponding oxides which form a three-dimensional network, the amorphous glass matrix of SiO2/B2O3/Al2O3 with metal ions occupying interstitial sites. In addition to the above-captioned alkaline and alkaline earth metal ions, transition metal ions as e.g. Zn2+ and Zr2+ may be incorporated in the matrix as network modifying agents.
  • [0057]
    In another embodiment of the invention, the glass may be produced with methods known in the state of the art by the melting of the raw material SiO2 and of carbonates of the alkali or alkaline earth metals Na2CO3, K2CO3 or CaCO3.
  • [0058]
    The reaction may be described as follows:
  • [0059]
    Na2CO3+SiO2→Na2SiO3 (=Na2O.SiO2)+CO2
  • [0060]
    K2CO3+SiO2→K2SiO3 (=K2O.SiO2)+CO2
  • [0061]
    CaCO3+SiO2→CaSiO3 (=CaO.SiO2)+CO2
  • [0062]
    However in most cases, there is no pure silicate matrix but a borate-aluminate-silicate-matrix i.e. regarding the network building constituent a part of SiO2 is substituted by B2O3 and Al2O3).
  • [0063]
    The sol:pigment ratio has a considerable effect on the yield of magnetic particles provided by this invention. It is essential for the process that the sol can still be pumped and sprayed which is in the skill of artisan.
  • [0064]
    To create a powder, the slurry is preferably sprayed through a two-fluid nozzle as described in FIG. 1 and in Example 1.3. Suitable spray-drying systems are produced by Nubilosa Molekularzerstäubung, Ladisch GmbH & Co. KG, Konstanz, Germany, e.g. the “Labor-Zerstäubungstrockner (Typ LTK)” or by Büchi A G, Uster, Switzerland, e.g. the Mini Spray Dryer (Type B-191).
  • [0065]
    Because of the diameter ratios of magnetic cores to the glass shell are less than 1 to 10, preferably between 1:10 and 1:1000, the geometry and the number of incorporated magnetic cores or of their inert carriers do not determine shape and size of the particles but the conditions of manufacturing, in particular the conditions during spray drying. In other words, the choice of pressure, inlet temperature, outlet temperature and flow rate during the spray drying procedure are the degrees of freedom which will determine the size distribution, the shape of the glass drops and thereby will modify the MGPs. When the spray pressure is increased, the size distribution will shift into the sub-μ-range. The decreased temperature of the spray drying process will lead to slower evaporation of the solvent and thereby the form of the MGPs will come closer to an ideal sphere, i.e. the ratio of the radii in xy- and xz-plane will become approximately 1. The ratio of the radii will vary between 0.8 and 1.2, preferably between 0.9 and 1.1.
  • [0066]
    In a preferred embodiment of the invention the nozzles are heated. The inlet temperature is between 120° C. and 500° C., preferably between 170° C. and 230° C. or 150° C. and 230° C., most preferably between 150° C. and 200° C. or 190° C. and 210° C. or at 200° C. or slightly less. The outlet temperature depends on the boiling point of the sol and thereby on the solvent and may be above, equal or slightly under, i.e. less than 10° C., the boiling point of the solvent. When ethanol is used as solvent, it is between 50° C. and 300° C., preferably 70° C. and 150° C., most preferably between 80° C. and 110° C. The optimal temperature is between 90° C. to 100° C. The nozzle pressure is more than 3 bar, preferably it is regulated to 4 to 6 bar. The artisan will appreciate the fact that the exact parameters will depend on the spray-drying system used. However, he can transfer the teachings of the present invention to any other spray-drying and find out the parameters by taking the disclosures of this invention into account. Formula as described in Masters: Spray Drying Handbook, Fifth Edition, John Wiley & Sons, 1991, New York can lead him the way to find out which parameters have to be chosen for another setting. Preferably, he will question the manuals of his spray-drying system or contact the technical service of the spray-drying system manufacturer.
  • [0067]
    To optimize the yield, the densification or sinter temperature should be as high as possible, i.e. slightly below the melting range. If it is too high, however, the particles will stick together and form agglomerates that must be sieved out. If too low, the MGPs will not be optimally densified. Additional treatment of the particles at too high temperature will result in a loss of magnetic properties. Too high temperatures should therefore be omitted. The exact temperatures depend on the glass composition but may be between 400° C. to 1200° C. In the case of the EJ glass composition the sinter temperature is between 720° C. and 770° C., preferably around 750° C. It is in the skill of the artisan to find out the temperatures for each glass composition when taking the teachings of the present invention into account. According to the present invention, the spray-dried MGP powder may be further processed as depicted in FIG. 2 and described in Example 1.4. Preferably, the powder is heated for 1 hour to 200° C., optionally cooled to room temperature and heated to 750° C. (densification or sinter temperature) in a nitrogen atmosphere with a heating rate of 1 K/min and is held at that temperature for 1 hour. Then the furnace is cooled to 150° C. and heated again to 200° C. for one hour in air. After the cooling to room temperature, the powder is transferred to a sieve (50 μm) and sieved for 30 min. The sieved sample is bottled and sterilized at 200° C. for 4 h and then cooled to 80° C. Then the glass vessels are taken from the oven, covered with sterile foil and closed.
  • [0068]
    Surprisingly, the magnetic particles provided by the invention are especially suited for isolating biological materials from samples. In addition, the core material is a natural resource and therefore causes little ecological concern. Moreover, the particles according to the invention are inexpensive and easy to manufacture.
  • [0069]
    Another object of the invention is a procedure for isolating a biological material by bringing a sample containing the biological material in a liquid in contact with the magnetic particles according to the invention under conditions in which the biological material binds to the particle surface, and separating the biological material from the liquid. According to the invention the term “in a liquid” means that liquid may be added to the sample before the magnetic particles are added. However, it shall also comprise the situation when the sample itself has a low viscosity and is itself a liquid so that no additional liquid or buffer may have to be added to the sample for the sample preparation which may be done by adding solid agents before the magnetic glass particles are added. The order of reagent addition may be varied according to process requirements.
  • [0070]
    Biological materials are understood to mean materials with a particular or molecular basis. They include, in particular, cells such as viruses or bacteria, as well as isolated cells from multicellular organisms as e.g. human and animal cells such as leucocytes, and immunologically active low and high molecular chemical compounds such as haptens, antigens, antibodies and nucleic acids. Nucleic acids such as DNA or RNA are especially preferred. In one embodiment of the invention mixtures of specific nucleic acids are purified, in which the target nucleic acid(s) may be a minor component in terms of concentration (or may be present in low abundance). According to the present invention, a target nucleic acid shall be the nucleic acid of interest, i.e. a nucleic acid which shall be investigated as its presence is indicative of a certain condition or disease of a human or animal. For example, the presence of a viral sequence (e.g. from Hepatitis B Virus, Hepatitis C Virus or Human immunodeficiency virus) indicates that the respective individual is infected by the specific virus. Then, this viral sequence would be the target sequence. Other target sequences are sequences which are indicative of a predisposition of an individual to a certain disease as e.g. an inherited disease as sickle cell anemia or to certain types of cancer. This examples should be illustrative of the invention but should not ne delimiting.
  • [0071]
    Samples according to the invention include clinical samples such as blood, serum, oral rinses, urine, cerebral fluid, sputum, stool, biopsy specimens and bone marrow samples. The sample can also be of a type used for environmental analysis, food analysis or molecular biology research, e.g., from bacterial cultures, phage lysates and products of amplification procedures such as the PCR.
  • [0072]
    The procedure described can be used to isolate native or modified biological material. Native biological material is understood to be material, the structure of which was not irreversibly changed compared with the naturally-occurring biological materials. This does not mean that other components of the sample can not be modified, however. Modified biological materials include materials that do not occur in nature, e.g., nucleic acids that are modified by attaching to them groups that are reactive, detectable or capable of immobilization. An example of this are biotinylated nucleic acids. In certain cases the sample can be used without pretreatment in the isolation procedure according to the invention. In many cases, however, the sample should be lysed using an appropriate method, releasing the biological material contained in the sample. Procedures for lysing samples are known by the expert and can be chemical, enzymatic or physical in nature. A combination of these procedures is applicable as well. For instance, lysis can be performed using ultrasound, high pressure, by shear forces, using alkali, detergents or chaotropic saline solutions, or by means of proteinases or lipases. With regard for the lysis procedure to obtain nucleic acids, special reference is made to Sambrook et al.: Molecular Cloning, A Laboratory Manual, 2nd Addition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y. and Ausubel et al.: Current Protocols in Molecular Biology 1987, J. Wiley and Sons, NY.
  • [0073]
    In addition to the biological material to be isolated, the sample can also contain other components in a liquid such as cell residue, proteins, salts and other substances that are not to be isolated. This sample, which preferably contains the biological material in native form, is brought in contact with the particles under conditions in which the target biological material binds to the particle surface. The conditions for this depend on the type of biological material involved, but are basically known. They also depend on the method by which the biological material is bound to the surface. If immunological interactions are utilized for the binding, for instance, conditions must be selected that are suitable for the formation of immunocomplexes. If modified nucleic acids are used, the binding can take place via the groups of nucleic acids that represent the modification, e.g., biotin via binding with streptavidin-coated surfaces. With nucleic acids in particular, however, a direct binding of nucleic acids to glass is preferred because among other reasons the nucleic acids do not have to be modified and even native nucleic acids can be bound. The procedure for binding native nucleic acids to glass particles can be analogous to the procedure described in the prior art. It is preferably performed in the presence of chaotropic salts with a concentration of between 2 and 8 mol/l, and preferably between 4 and 6 mol/l. Chaotropic salts can be sodium iodide, sodium perchlorate, guanidinium thiocyanate, guanidinium isothiocyanate or guanidinium hydrochlorite. Other compounds are also possible.
  • [0074]
    To bring the sample in contact with the particles, the sample is mixed with the particles and incubated for a period of time sufficient for the binding to occur. Experts are usually familiar with the duration of the incubation step from procedures for performing treatment with non-magnetic particles. This step can be optimized by determining the quantity of immobilized biological material on the surface at different points in time. Incubation times of between 10 seconds and 30 minutes can be appropriate for nucleic acids.
  • [0075]
    Depending on the size and type of magnetic particles, the particles either separate out of the fluid during the incubation period itself or the suspension remains intact for a longer period of time. If the particles are very small and unmagnetized, the suspension remains intact for a longer period of time. If the particles are of larger size, the particles slowly separate out of the fluid during the incubation period.
  • [0076]
    Immobilization is preferably not performed via precipitation by lowering the solubility of the materials to be immobilized. Rather, immobilization is based on biospecific interactions (capture molecules) or adsorption. This largely prevents contaminants from being non-specifically included.
  • [0077]
    After incubation, the biological material is separated from the liquid. This is achieved in general by separating the material bound to the magnetic particles by applying a magnetic field. For instance, the magnetic particles can be pulled to the wall of the vessel in which incubation was performed. The liquid containing the sample contents that were not bound to the magnetic particles can then be removed. The removal procedure used depends on the type of vessel in which incubation was performed. Suitable steps include removing the liquid via pipetting or aspiration.
  • [0078]
    The magnetic particles can then be purified one or more times using a wash solution, if desired. A wash solution is used that does not cause the biological material to be released from the particle surface but that washes away the undesired contaminants as thoroughly as possible. This wash step preferably takes place by incubating the wash solution with the particles. The particles are preferable resuspended during this step, e.g., by means of shaking or applying a magnetic field that is not identical to the first magnetic field. The contaminated wash solution is preferably separated just like the sample in the step described above for binding the biological material.
  • [0079]
    After the last wash step, the magnetic particles can be dried briefly in a vacuum, or the fluid can be allowed to evaporate. A pretreatment step using acetone may also be performed. If desired, the biological material purified in this manner can be separated from the magnetic particles. This step also depends on the manner in which the biological material was bound to the magnetic particles. If the biological material is native nucleic acids and the magnetic particles are glass-coated particles, the nucleic acids can be removed from the particles according to the invention using an elution buffer having a low salt content. Buffers of this nature are known from DE 3724442 and Analytical Biochemistry 175, 196-201 (1988). The elution buffers with a low salt content are in particular buffers with a content of less than 0.2 mol/l. In an especially preferred embodiment, the elution buffer contains Tris. In another special embodiment, the elution buffer is demineralized water.
  • [0080]
    In yet another embodiment, the purification and isolation procedure described is performed after the cells (e.g., viral particles or prokaryotic or eukaryotic cells) are separated immunomagnetically from a bodily fluid or tissue. In this step, the sample is incubated, e.g., while shaking, with magnetic particles to which an antibody against an antigen on the cell is immobilized. These particles can be particles according to the invention or commercially available particles (e.g., MACS Microbeads from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). After a magnetic field is applied, one or more wash steps are performed using a saline solution. Particles are obtained to which the desired cells are bound. The bound cells are then resuspended in a saline buffer. In a preferred embodiment, this saline buffer is a chaotropic saline solution so that the nucleic acids contained in the cell are released from the cells.
  • [0081]
    An especially advantageous procedure for isolating nucleic acids from samples containing cells is achieved by combining the isolation of cells described above with the isolation of nucleic acids also described above, on the magnetic particles according to the invention. The advantage of this embodiment is its potential simplicity (single-tube method), high sensitivity (especially important in medical microbiology and oncology), and the ease with which it can be automated.
  • [0082]
    The biological materials isolated using the procedure according to the invention can now be used further as necessary. For instance, they can be used as a substrate for various enzymatic reactions. When nucleic acids are involved, they can be used for sequencing, radioactive or non-radioactive labelling, amplification of one or more of the sequences they contain, transcription, hybridization with labelled probe nucleic acids, translation or ligation. An advantage of the procedure according to the invention is that it is very easy to separate the biological material from the fluid. In the prior art, a centrifugation step was used to separate the glass particles from contaminants, or, when the biological material is bound to glass fiber filters, the fluid is drawn through the filters. This is a limiting step that makes it difficult to process large quantities of sample. The biological materials can be separated from contaminants more effectively using the particles according to the invention. In particular, inhibitors for certain enzymatic reactions can be removed to a large extent according to the invention.
  • [0083]
    In the preferred embodiment, the particles according to the invention are added to the lysis mixture. After a suitable period of time for adsorption to take place—which can be optimized by mechanical agitation—the particles are separated from the surrounding fluid that contains additional cell components that are not to be detected.
  • [0084]
    This is performed preferably by applying a magnetic field by placing a magnet against the vessel wall.
  • [0085]
    To remove any contaminants that may still be present, a wash step is preferably performed with a fluid that does not cause the nucleic acids to be determined to be released from the glass surface. An elution buffer having reagent conditions under which the nucleic acids separate from the glass surface is added to remove the nucleic acids from the glass surface. These conditions are low salt conditions in particular. Depending on the intended further use of the nucleic acids, the fluid can now be separated from the particles and processed further. This separation step is preferably performed via application of a magnetic field so that the particles are separated from the eluate.
  • [0086]
    A preferred embodiment of the present invention is to use the MGPs of the present invention in automatable methods as e.g. described in WO 99/16781. Automatable method means that the steps of the method are suitable to be carried out with an apparatus or machine capable of operating with little or no external control or influence by a human being. Automatized method means that the steps of the method are carried out with an apparatus or machine capable of operating with little or no external control or influence by a human being. Only the preparation steps for the method may have to be done by hand, e.g. the storage containers have to filled up and put into place, the choice of the samples has to be done by a human being and further steps known to the expert in the field, e.g. the operation of the controlling computer. The apparatus or machine may e.g. add automatically liquids, mix the samples or carry out incubation steps at specific temperatures. Typically, such a machine or apparatus is a robot controlled by a computer which carries out a program in which the single steps and commands are specified. Preferred automatic methods are those which are carried out in a high-throughput format which means that the methods and the used machine or apparatus are optimized for a high-throughput of samples in a short time.
  • [0087]
    In another embodiment the MGPs according to the present invention are used in semi-automated process which means that some reaction steps may have to be done manually. In a preferred embodiment of the invention, a suspension containing MGPs according to the present invention is taken from a storage container and partial volumes are added to different reaction vessels. Reaction vessels may be reaction tubes made from plastics eventually in mictrotitreplate format contain 96 or 384 or more wells where a reaction can be carried out. However, these vessels may be made from other material e.g. from steel.
  • [0088]
    A preferred embodiment of the invention are purification methods followed by a detection step or purification methods followed by an amplification and detection step. The target nucleic or nucleic acid or nucleic acids of interest may be contained in a matrix of non-target nucleic acids, and may even be a minor component in said mixture of specific nucleic acids. Suitable DNA detection methods are known to the expert in the field and are described in standard textbooks as Sambrook et al.: Molecular Cloning, A Laboratory Manual, 2nd Addition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y. and Ausubel et al.: Current Protocols in Molecular Biology 1987, J. Wiley and Sons, NY. There may be also further purification steps before the DNA detection step is carried out as e.g. a precipitation step. The detection methods may include but are not limited to the binding or intercalating of specific dyes as ethidiumbromide which intercalates into the double-stranded DNA and changes its fluorescence thereafter. The purified DNA may also be separated by electrophoretic methods optionally after a restriction digest and visualized thereafter. There are also probe-based assays which make use of a oligonucleotide hybridisation to specific sequences and following detection of the hybrid. It is also possible to sequence the DNA after further steps known to the expert in the field. Newer methods apply a diversity of DNA sequences to a silicon chip to which specific probes are bound and yield a signal when a complementary sequences bind.
  • [0089]
    Preferred methods according to the invention are amplification methods as the ligase chain reaction and the polymerase chain reaction which specifically amplify target sequences to detectable amounts. Particularly preferred detection methods are the TaqMan® method disclosed in WO92/02638 and the corresponding US patents U.S. Pat. No. 5,210,015, U.S. Pat. No. 5,804,375, U.S. Pat. No. 5,487,972. This method makes use of the exonuclease activity of a polymerase to generate a signal. In detail, the nucleic acid is detected by a process comprising contacting the sample with an oligonucleotide containing a sequence complementary to a region of the target nucleic acid and a labeled oligonucleotide containing a sequence complementary to a second region of the same target nucleic acid sequence strand, but not including the nucleic acid sequence defined by the first oligonucleotide, to create a mixture of duplexes during hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the first oligonucleotide and to the labeled oligonucleotide such that the 3′-end of the first oligonucleotide is adjacent to the 5′-end of the labeled oligonucleotide. Then this mixture is treated with a template-dependent nucleic acid polymerase having a 5′ to 3′ nuclease activity under conditions sufficient to permit the 5′ to 3′ nuclease activity of the polymerase to cleave the annealed, labeled oligonucleotide and release labeled fragments; and the signal generated by the hydrolysis of the labeled oligonucleotide is detected and/or measured. TaqMan® technology eliminates the need for a solid phase bound reaction complex to be formed and made detectable.
  • [0090]
    In more general terms, a procedure for the purification of biological material followed by a detection step is disclosed wherein the amplification and/or detection reaction is a homogeneous solution-phase multiplex assay for the simultaneous detection of multiple targets (see examples 7.2).
  • [0091]
    In another embodiment of the invention, the described purification procedure is combined with a amplification procedure using one of the methods described in the following, preferably the use of blocking oligonucleotides. A problem often associated with amplification especially of low amounts of target nucleic acid is the activity of thermostable polymerases at lower temperatures (room temperature up to 40° C.). At this temperature primer oligonucleotides often bind unspecifically to each other or to background nucleic acid and may be extended by the polymerase. This entails a decrease of reaction components as well as to a higher level of background signal and consequently to a decreased sensitivity. It may also lead to false-positive results. In order to avoid unspecific activity of polymerases several approaches have been described, like “hot start”-PCR (Chou et al., 1992, Nucl. Acid Res 20, 1717-1723), covalently modified polymerases (e.g. AmpliTaq Gold, Perkin Elmer) or antibodies (Scalice et al., J. Immunol. Methods, 172, 147-163, 1994) and oligonucleotides (Dang and Jayasena, JMB 264, 268-278, 1996; U.S. Pat. No. 5,763,173; U.S. Pat. No. 5,693,502). According to the present invention blocking oligonucleotides shall be oligonucleotides which are able to block the active center of the polymerases up to the said temperature. These oligonucleotides may be e.g. an aptamer as described in Example 7.2.2.1.3. In another embodiment of the invention aptamers (see e.g. in Example 7.2.2.1.3) and/or modified primers alone (see e.g. in Example 7.2.2.1.3) can be used in an amplification reaction and the detection methods connected thereto. This has advantageous effects and can be considered to be an invention on its own which provides superior results. In a preferred embodiment of the invention the 3′-terminal nucleobase, preferably an adenin is modified with a p-(t-butyl)-benzyl-residue. Further modifications, including those at the 3′-1-position are described in EP 866 071 A2 which is incorporated herein by reference.
  • [0092]
    In still another embodiment of the invention for the purification of biological material followed by a detection step is disclosed wherein for at least 5 cycles of the polymerase chain reaction, the annealing temperature is less than 8° C., preferrably less than 3° C. above the dissociation temperature of the polymerase-aptamer complex.
  • [0093]
    The following examples serve to illustrate the embodiments of the invention.
  • FIGURE LEGENDS
  • [0094]
    [0094]FIG. 1: Flow scheme for the production by sol synthesis & spray drying:
  • [0095]
    [0095]FIG. 2: Flow scheme for the preparation of the raw MGPs
  • [0096]
    [0096]FIG. 3: Schematic depiction of the spray dryer manufactured by Nubilosa. The details are described in the text under Example 1.3
  • [0097]
    [0097]FIG. 4: High resolution scanning electron microscopic image of MGP with Merck pigment BM
  • [0098]
    [0098]FIG. 5: High resolution scanning electron microscopic image of MGP with CERAC pigment
  • [0099]
    [0099]FIG. 6: High resolution scanning electron microscopic image of MGP with Merck pigment MMB
  • [0100]
    [0100]FIG. 7: High resolution scanning electron microscopic image of MGP with Strem pigment
  • [0101]
    [0101]FIG. 8: High resolution scanning electron microscopic image of MGP with BASF pigment FA
  • [0102]
    [0102]FIG. 9: High resolution scanning electron microscopic image of MGP with BASF pigment CE-HQ
  • [0103]
    [0103]FIG. 10: High resolution scanning electron microscopic image of MGP with BASF pigment CE-SU
  • [0104]
    [0104]FIG. 11: Influence of the magnetic core pigment or the spray dryer on the RNA isolation (spray dryer: Büchi (B) or Nubilosa (N))
  • [0105]
    [0105]FIG. 12: Influence of the parameter “Spray pressure” on the isolation of DNA or RNA
  • [0106]
    [0106]FIG. 13: Influence of different MGP production parameters on the isolation of RNA (spray pressure, (L=air, N=nitrogen), inlet temperature (E) or outlet temperature (A)
  • [0107]
    [0107]FIGS. 14a, b: Sedimentation behaviour of different MGPs in different suspension media
  • [0108]
    [0108]FIG. 15: HREM-image of sample EJ0096.5R-01; MGP with CERAC pigment
  • [0109]
    [0109]FIG. 16: HREM image of EJ0100.5R-01, sprayed at low pressure and high temperature, consisting mainly of deformed μ-scale particles
  • 1 EXAMPLE 1 Production of the Magnetic Glass Particles
  • [0110]
    Classically, raw materials like SiO2 and alkali- or alkaline earth carbonates (Na2CO3, K2CO3, CaCO3) are molten together
  • [0111]
    type of reaction:
  • [0112]
    Na2CO3+SiO2→Na2SiO3 (=Na2O.SiO2)+CO2
  • [0113]
    K2CO3+SiO2→K2SiO3 (=K2O.SiO2)+CO2
  • [0114]
    CaCO3+SiO2→CaSiO3 (=CaO.SiO2)+CO2
  • [0115]
    In most cases, however, a composite matrix of silicate, borate and aluminate is used, i.e. pertinent to the matrix constituents, SiO2 is partially replaced by B2O3 and Al2O3.
  • [0116]
    Alternatively, glass can be synthesized via sol-gel-reaction.
  • [0117]
    type of reaction:
  • [0118]
    either acid-catalyzed sol-gel reaction, e.g.
  • [0119]
    BCl3+3H2O→B(OH)3+3H(+)Cl(−)
  • [0120]
    AlCl3+3H2O→Al(OH)3+3H(+)Cl(−)
  • [0121]
    SiCl4+4H2O→Si(OH)4+4H(+)Cl(−)
  • [0122]
    Na(+)NO3 (−)+H2O→Na(+)OH(−)+H(+)NO3 (−)
  • [0123]
    K(+)(−)OOCCH3+H2O→K(+)OH(−)+H(+)(−)OOCCH3
  • [0124]
    or base-catalyzed, as in our case, e.g.
  • [0125]
    K(+)(−)OCH2CH3+H2O→K(+)OH(−)+HOCH2CH3
  • [0126]
    Na(+)(−)OCH3+H2O→Na(+)OH(−)+HOCH3 Al (3+)[(−)OCH2CH2CH3]3+3H2O→Al(OH)3+3HOCH2CH2CH3 B(OCH 2CH3)3+3H2O→B(OH)3+3HOCH2CH3
  • [0127]
    Advantage: alcohols are easily evaporated during spray-drying; ideally no recristallisation of salts on the surface
  • [0128]
    Thus, alcoholates are turned into hydroxides, which yield by way of elimination of water the corresponding oxides. These, then, form a 3-dimensional, amorphous glass matrix consisting of SiO2/B2O3/Al2O3, into which certain metal oxide ingredients are embedded as matrix bond separators, e.g.
  • [0129]
    For the experiments described here, the glass component was produced via base-catalyzed sol-gel synthesis. The glass composition used in all experiments was (unless stated otherwise) as follows:
  • [0130]
    70.67 Mol % SiO2, 14.33 Mol % B2O3, 5.00 Mol % Al2O3, 4.00 Mol % K2O, 2.00 Mol % CaO, 4.00 Mol % ZnO
  • [0131]
    (calculated from the mass of particular educts brought to reaction)
  • [0132]
    1.1 Description of the Investigated Magnetic Pigments
  • [0133]
    Different types of magnetic pigments were investigated and are summarized in Table 1.
  • [0134]
    1.2 Production of the Coating Sol (EJ-Composition)
  • [0135]
    The educts are added in the following order and amount into the heatable stirring vessel:
    Tetraethoxysilane (TEOS)  10700 ml
    Triethylborate (TEB)  3305 ml
    K-methanolate 25% (W/V) in Methanol   1601 ml
    Ethanol  11292 ml
    Aluminiumisopropanolate   1385 g
    Calcium  54.4 g
    Zincacetate   498 g
  • [0136]
    The vessel is closed thereafter. The sol is heated to 70° C. and stirred overnight (15 h). The temperature is regulated via a thermosensor which dips into the liquid.
  • [0137]
    Then the sol is heated to 90° C. and the alcohol/water mixture (Ethanol: 3781 ml, H2O: 1512 ml) is added at a rate of 5000 ml/h. The vessel is cooled to 20° C. after the complete addition of the said mixture.
  • [0138]
    The lid is opened and 10249 g magnetic pigment (CERAC) is added under vigorous stirring. The prepared sol is transferred into the spray dryer via a hose.
  • [0139]
    1.3 Spray Drying
  • [0140]
    The inlet temperature of the spray dryer is regulated to 200° C. The nozzle pressure is regulated to 6 bar and the nozzle is cooled for 3 min with ethanol. Then the hose is connected to the outlet of the glass container and the pigment-containing sol is pumped at a rate of 110 ml/min to the two fluid nozzle (diameter of the opening: 2 mm; supplier: Nubilosa, Type 1B1VVS1) of the spray dryer via a ultrasound device (200 W). The spray drying system is shown schematically in FIG. 3. The particles which are formed in the first two minutes are discarded. After the complete spraying of the sol the container under the cyclone (AVO, see FIG. 3) with the particles will be taken and the particles will be further treated as described in the next steps.
  • [0141]
    The pigment containing sol is stirred in the stirring vessel to prevent sedimentation of the suspended particles. The sol is transferred from the vessel to the nozzle using a pump (SP). Nitrogen heated by an electric heater (EWT) is used as drying gas. The coated pigment is transferred from the drying chamber (T) to the cyclone (ZY). The dried powder an be removed from the container (AVO) under the cyclone. Very fine particles are removed by a filter (SF) from the nitrogen. The spray dryer is used with overpressure to prevent an air intake into the dryer. The gas flow is produced by gas blower (AV).
  • [0142]
    1.4 Further Processing of the Spray-Dried Powder
  • [0143]
    The powder is transferred into a ceramics bowl and heated in an furnace to 200° C. at a heating rate of 1 K/min. Then temperature is held at 200° C. for one hour and the oven is cooled to room temperature afterwards. The bowl is transferred to an atmosphere furnace with a volume of 27 l which is rinsed with 60 l nitrogen per hour. The furnace is heated to 750° C. with a heating rate of 1 K/min and is held at that temperature for 1 hour. Then the furnace is cooled to 150° C. and rinsed with 60 l air per hour. The furnace is heated to 200° C. with a heating rate of 2 K/min and is held at that temperature for 1 hour and is cooled to temperature afterwards. The powder is transferred to a sieve (50 μm) and sieved for 30 min. Afterwards, the sieved sample is bottled in glass containers, which are sterilized unclosed in an oven which is heated to 200° C. with a heating rate of 1 K/min, held at that temperature for 4 h and then cooled to 80° C. Then the glass vessels are taken from the furnace, covered with sterile foil and closed with a lid.
  • [0144]
    A summary of the important process parameters is presented in Table 2 and Table 3, the produced individual samples with some data are provided in Table 3. Each sample has a specific code. The first two letters describe the used glass chemistry (see below) and the next four numbers encode the production process (see Table 2). The number after the describes the used pigment (see Table 1). The letter R means that the fine content has not been removed, whereas E means that the fine content has been removed with ethanol. The last two numbers describe the number of the lot. The composition of the glass (code EJ) as calculated from the quantity of the educts was 70.67 Mol % SiO2, 14.33 Mol % B2O3, 5.00 Mol % Al2O3, 4.00 Mol % K2O, 2.00 Mol % CaO, 4.00 Mol % ZnO. The composition of the glass (code RN) was 74 Mol % SiO2, 15 Mol % B2O3, 5.00 Mol % Al2O3, 4.00 Mol % K2O, 2.00 Mol % CaO. The composition of the glass (code EP) was 73.61 Mol % SiO2, 14.93 Mol % B2O3, 5.21 Mol % Al2O3, 4.17 Mol % K2O, 2.08 Mol % CaO.
  • 2 EXAMPLE 2 High Resolution Scanning Electron Microscopy
  • [0145]
    To obtain information about the surface, size and form of the MGP, we carried out investigations with a high resolution scanning electron microscope manufactured by the company JEOL (JSM). The samples were spread onto the sample holder with an electrically conducting double-sided adhesive and sputtered with gold for 36 s with a current of 30 mA. For the imagining of the surface, the emitted secondary electrons (topography) as well as the back-scattered electrons (order number contrast) were viewed. The used primary electron voltage was 10 kV (secondary electrons) or 25 kV (back-scattered electrons).
  • [0146]
    The FIG. 4 shows MGPs with mica covered with ferric oxide as magnet pigment (BM). The fissure in the glass shell can be clearly seen which stem from the spray drying of the particles when the layer starts to shrink on a non-shrinking substrate (drying fissures). Further more a greater number of spheric particles can be seen which do not contain one of the bigger magnet particles (10-60 μm). These spheres without magnetic core (non-magnetic fine content) cannot be removed in a magnetic field, may be transferred into the PCR reaction and will interfere with this reaction.
  • [0147]
    The particles with CERAC pigment (see FIG. 5) do not have fissure, as the magnet pigment (23 nm average particle size) does not cause tension forces in the layer so that no fissures are created during the spray drying process. Furthermore, the small CERAC-particles are also incorporated in the fine content so that there are no non-magnetic particles contained therein. The particles are also substantially smaller than the particles with mica which can be observed in these figures with the same magnification. MGPs with the other magnetic pigments are shown at the same magnification in FIG. 6 to FIG. 10.
  • 3 EXAMPLE 3 Physical Investigations of the MGPs
  • [0148]
    To allow an evaluation of the MGPs before the actual functional tests, in addition the HREM investigations further physical measurements were done with the MGPs. It was interesting to learn about the iron solubility in water, the magnetic force, the fine content and the density. In the following the experiments are described and the results presented.
  • [0149]
    3.1 Extinction Measurements
  • [0150]
    To test whether the fines have been removed, 1000 mg of the temperature-treated and sieved powder, are weighed into a 50 ml centrifugation tube (Sarstedt), 40 ml water added and dispersed by shaking. Then, the tube is dipped into a ultrasonic bath (Sonorex (RK 102H; 120/240W) with the total filling height and is put into a magnetic separator (Roche Diagnostics GmbH RD Art.Nr. 1858 025). After 3.5 min magnetic separation, liquid is taken from the vessel with a pasteur glass pipette in the height of the 15 ml mark at the opposite side of the magnet and filled into a 5 mm quartz cuvette (Type 110-QS, Hellma). The extinction of the supernatant is measured in an UV-VIS-NIR spectrometer (Hitachi U-3000) against a corresponding cuvette filled with deionized water. The measuring range was 200 to 1100 nm in steps of 1 nm to investigate this wavelength range for the absorption bands of eventual impurities. The extinction at a wavelength of 280 m and 400 nm was taken as a reference.
  • [0151]
    3.2 Density Measurements
  • [0152]
    For the density measurements a gas pyknometer (AccuPyc 1330, Fa. Micromeritics) is used. Helium 6.0 is used as gas. The device is calibrated with the supplied standard (steel beads with known volume). Further, the sample is dried for at least 1 hour at 150° C. and then filled into the measurement container and weighed. After the measuring container is put into the gas pyknometer, 10 washing cycles and 5 measuring cycles are used to determine the average value and the standard deviation.
  • [0153]
    3.3 Determination of the Iron Solubility
  • [0154]
    The iron solubility of the coated and temperature-treated samples is determined with Inductive Coupled Plasma—Atomic Emission Spectroscopy (JY 24, company ISA). Therefor, 1 g of the sample is transferred into a 50 ml polypropylene-tube, filled up with distilled water and kept at a temperature of 60° C. for 20 hours. Then, the samples are filtrated with a 0.2 μm syringe filter and the filtrate is measured. Four single determinations are carried out at a wavelength of 259,940 nm and an average value is computed therefrom.
  • [0155]
    3.4 Measurement of the Magnetic Force
  • [0156]
    For the measurement of the magnetic force a PP-weighing tube (Company Licefa, Art. Nr. V2-3) completely filled with MGPs is weighed. With the help of a stencil, this sample container is placed in the middle of LDPE tube (Company Kartell, TS 735) burdened with brass so that the lid of the tube can be still closed. The vessel is positioned in the middle of a balance (detection accuracy 0.1 g) with the help of another stencil. After the equalization of the balance, a plastics cap which contains an cylinder-shaped magnet (diameter: 30 mm; height: 113.5 mm; material: samarium-cobalt 2/17) is positioned above the scale.
  • [0157]
    Thereby, the MGPs in the scale are attracted against to the gravity thereby decreasing their weight. After the adjustment of the equilibrium (1.5 min) the weight loss of the sample is determined and the value is normalized for 250 mg MGP.
  • [0158]
    3.5 Results
  • [0159]
    The results of the physical characterization of the MGPs with EJ composition and different pigments are summarized in Table 3. It is noticeable that the extinction values are very low when CERAC pigment is used. This can be explained by the lack of non-magnetic fine content because the small CERAC pigment (23 nm) can be incorporated into each magnetic glass particle. The density of the CERAC-MGPs is not very high. This has a positive influence on the sedimentation velocity. The magnetic force is subjected to high fluctuations, however, it is positively influenced by the use of CERAC-pigments. The CERAC-MGPs have a comparatively high leak of iron to the water. However, even ten-fold higher values did not show any effect on the PCR process.
  • [0160]
    In summary, it can be said that the physical properties do not have any significant differences between the different pigments but the CERAC-MGPs have a very low fine content.
  • 4 EXAMPLE 4 Influence of the Magnetic Core Pigment and of the Spray Dryer on the BET Surface
  • [0161]
    The surfaces were determined using a device from Micromeritics (Type ASAP 2400). The measurement was performed at liquid nitrogen temperature. Nitrogen 5.0 was used as measuring gas and Helium 4.6 was used as inert gas. Typically, 5 g of sample was used for one measurement.
  • [0162]
    The surface of the MGPs plays an obvious role for the isolation of DNA and RNA. The higher the surface the more DNA can be bound by the same mass of MGPs. It is also possible to use less MGPs and obtain the same performance. This has the effect that the intergrain volume is less which means that less alcohol may be introduced as contaminant into the PCR reaction. The data for the measured BET-surfaces for some samples are summarized in Table 4. When the surfaces of the samples produced on the big spray dryer are compared, it should be noted that the samples with small pigments (BASF FA, STREM and CERAC) have relatively high surfaces, whereas the big Merck pigments have small surfaces. This may be caused by the fact that big magnetic particles cause big MGPs whereas the small magnetic particles are incorporated into the spherical droplets during the spray drying process and have therefore similar surfaces under similar spraying conditions. The BASF CE-SU particles have a size distribution in between the small (BASF FA, STREM and CERAC) and the big Merck particles. In contrast to the Merck particles, they do not possess a structured surface so that these particles have a very smooth glass surface. This results into a smaller surface. A higher pressure was used with the smaller spray dryer and the nozzle of this spray dryer is remarkably smaller than that of the bigger spray dryer. This causes smaller particles whereby the surface increases remarkably. This is in good agreement with the results of Example 5.2.1
  • [0163]
    Any influence of the magnetic pigment or the spraying pressure on the results of further physical investigations cannot be recognized except the above-mentioned influence on the fissure formation and the fine content. However, only the measurements of the surface show a direct connection to the results of the functional investigations. Other physical investigation results do not show this direct correlation.
  • 5 EXAMPLE 5 Binding Studies via Radio Tracing
  • [0164]
    There are several methods for the evaluation of MGPs with regard to their suitability for the extraction of nucleic acids. The determination of the extinction at 260 nm before and after the purification is not very sensitive and does not resemble the situation when small amounts of target nucleic acid are extracted from clinical material. The results of functional evaluation methods which rely upon a method for the amplification of nucleic acids, as e.g. by PCR, RT-PCR, NASBA or anything similar, are often not enough convincing. Furthermore, these methods, which are suitable for the determination of a small number of copies of the target genome, are susceptible to disturbances by substances from the sample material or from the sample preparation process. Radioactive binding studies are a suitable analytical method which makes it possible to analyze the sample preparation process step by step. The performance data are no absolute data but are always relative to the performance of a reference particle
  • [0165]
    5.1 Experimental Protocol
  • [0166]
    At first, radioactively labelled DNA or RNA is synthesized enzymatically in a PCR or in vitro transcription process in the presence of 32P-markied desoxynucleoside or nucleoside-triphosphates. Then, the marked DNA or RNA is separated from the free nucleoside-triphosphates, the content determined and a defined dilution is prepared.
  • [0167]
    A small amount of marked DNA or RNA is added to each sample before the examination. During sample preparation, all nucleic acids bind to the MGPs in the presence of chaotropic agents. The MGPs are pelleted by the exertion of magnetic forces and the supernatants can be discarded. The pellet is washed and the bound nucleic acids are eluted at elevated temperature by the reversal of the reaction conditions, i.e. by the addition of a low salt elution buffer. After the binding and an optionally after the washing step, an aliquot of the particle supernatant is spotted onto a filter. The eluate as well as the in water redispersed MGP are spotted onto a filter as well and dried thereafter. At last, the filters are measured in a Beta.Counter and for each sample preparation a distribution is calculated.
  • [0168]
    5.1.1 Preparation of Radioactively labelled DNA
  • [0169]
    5.1.1.1 A.1.1. Reagents
  • [0170]
    Expand High Fidelity PCR System (Roche Molecular Biochemicals Cat. No. 732641)
  • [0171]
    dNTP Mix (Roche Molecular Biochemicals Cat. No. 1277049)
  • [0172]
    Deoxycytidine 5′-alpha-P32 Triphosphate dCTP 3000 Ci/mmol (Amersham Cat. No. PB 10205)
  • [0173]
    lambda-DNA (Roche Cat. No. 1029053) concentration 1 ng/ml
  • [0174]
    Radiotracer primer 1 (SEQ ID NO 1) concentration 5.3 OD260/ml
  • [0175]
    Radiotracer primer 2 (SEQ ID NO 2) concentration 5.2 OD260/ml
  • [0176]
    QIA Quick PCR Purification Kit (Qiagen Cat. No. 28104)
  • [0177]
    5.1.1.2 Reaction
  • [0178]
    29.51 μl double distilled water
  • [0179]
    5 μl Expand High Fidelity Buffer
  • [0180]
    2.5 l dNTP Mix (1:10) diluted with double distilled water
  • [0181]
    1 μl Radiotracer primer 1 (SEQ ID NO 1)
  • [0182]
    1 μl Radiotracer primer 2 (SEQ ID NO 2)
  • [0183]
    0.3 μl 32P-dCTP
  • [0184]
    10 μl lambda-DNA
  • [0185]
    0.75 μl Expand High Fidelity-Enzyme Mix
  • [0186]
    5.1.1.3 Amplification
  • [0187]
    2 min. 94° C.
  • [0188]
    10 cycles (10 sec. 94° C./30 sec. 60° C./60 sec. 72° C.)
  • [0189]
    20 cycles (10 sec. 94° C./30 sec. 60° C./60 sec. 72° C.+10 sec. 72° C. extension per cycle)
  • [0190]
    7 min 72° C.
  • [0191]
    4° C.
  • [0192]
    5.1.1.4 Purification
  • [0193]
    according to the QIA Quick PCR Purification Protocol (Qiagen)
  • [0194]
    5.1.1.5 Dilution
  • [0195]
    Dilution of DNA 1:10 in double distilled water and measurement in the Beta-Counter
  • [0196]
    5.1.2 A.2. Preparation of Radioactively Labelled RNA
  • [0197]
    5.1.2.1—Reagents
  • [0198]
    SP6/T7 Transcription Kit (Roche Molecular Biochemicals Cat. No. 999644)
  • [0199]
    Uridine 5′-alpha-P32 Triphosphate UTP 3000 Ci/mmol (Amersham Cat. No. PB 10203)
  • [0200]
    Plasmid pBKBH10S, linearized with EcoRI at 100 μg/ml
  • [0201]
    High Pure RNA Isolation Kit (Roche Molecular Biochemicals Cat. No. 1828665)
  • [0202]
    5.1.2.2—Reaction
  • [0203]
    2 μl 10× buffer
  • [0204]
    3 μl NTP Mix (AGC)
  • [0205]
    1 μl UTP (1:50 with double distilled water)
  • [0206]
    5 μl 32 P-UTP
  • [0207]
    7 μl linearized plasmid
  • [0208]
    1 μl RNAse Inhibitor (Roche Molecular Biochemicals Cat. No. 802808)
  • [0209]
    1 μT7 RNA-Polymerase
  • [0210]
    5.1.2.3 Transcription and DNAse-Digest
  • [0211]
    20 min incubation at 37° C.
  • [0212]
    Addition of 2 μl DNAse, RNAse free
  • [0213]
    10 min incubation at 37° C.
  • [0214]
    Addition of 178 μl double distilled water
  • [0215]
    5.1.2.4—Purification
  • [0216]
    according to the High Pure RNA Isolation Protocol (Roche Molecular Biochemicals)
  • [0217]
    5.1.2.5—Dilution:
  • [0218]
    Dilution of RNA 1:30 in double distilled water and measurement at the Beta-Counter
  • [0219]
    5.1.3—Radioactive Sample Preparation
  • [0220]
    5.1.3.1—Reagents
  • [0221]
    negative plasma
  • [0222]
    Proteinase K concentration 20 mg/ml (e.g. Roche Id.Nr. 1942387)
  • [0223]
    Poly-A-RNA (e.g. Roche Id.Nr. 108626) concentration 1 mg/ml; diluted 1:1000 (volume/volume ratio) with lysis buffer
  • [0224]
    lysis buffer (50 mM Tris pH 7.0, 15% (v/v) Polydocanol, SM Guanidiniumisothiocyanate, 1 mM DTT)
  • [0225]
    MGP (with glass composition EJ and different core pigments (BM, MMB, CERAC, STREM, BASF-FA, BASF-CE)) suspended in isopropanol at 60 mg/ml or at 6 mg/ml
  • [0226]
    Washing buffer (20 mM Tris pH 7.5, 20 mM NaCl, 70% (v/v) Ethanol)
  • [0227]
    Elution solution: double distilled water
  • [0228]
    Auxiliary material: Whatman GF/D Filter
  • [0229]
    5.1.3.2—Reaction (1.5 ml protocol)
  • [0230]
    80 μl Proteinase K
  • [0231]
    add 410 μl negative plasma and mix
  • [0232]
    add 500 μl lysis buffer and mix
  • [0233]
    add 10 μl radioactive labelled DNA or RNA and mix
  • [0234]
    10 min incubation at room temperature under shaking
  • [0235]
    add 500 μl MGP suspension (concentration 6 mg/ml) and mix
  • [0236]
    20 min. incubation at room temperature under shaking
  • [0237]
    separation for 2 min in Magnetic separator (Dynal)
  • [0238]
    discard supernatant and spot 300 μl onto a filter
  • [0239]
    add 750 μl wash buffer, vortex, 2 min separation
  • [0240]
    discard supernatant, eventually spot 375 μl supernatant onto a filter
  • [0241]
    repeat washing procedure twice
  • [0242]
    add 100 μl elution solution, incubate 5 min. at 80° C. in a thermomixer
  • [0243]
    separate 2 min in a magnet holder, spot supernatant onto a filter
  • [0244]
    add 100 μl elution solution, resuspend and spot MGPs onto a filter
  • [0245]
    dry filters for 60 min at 75° C. in a drying oven
  • [0246]
    transfer filter into scintillation tubes, add 5 ml scintillation solution and measure in the beta-counter
  • [0247]
    5.1.3.3—Experimental Protocol (1 ml protocol)
  • [0248]
    25 μl Proteinase K
  • [0249]
    add 415 μl negative plasma and mix
  • [0250]
    add 500 μl lysis buffer and mix
  • [0251]
    add 10 μl radioactive labelled DNA or RNA and mix
  • [0252]
    5 min incubation at room temperature under shaking
  • [0253]
    add 50 μl MGP suspension (concentration 60 mg/ml) and mix
  • [0254]
    20 min. incubation at room temperature under shaking
  • [0255]
    separation for 2 min in the magnetic separator (Dynal)
  • [0256]
    discard supernatant and spot 300 μl onto a filter
  • [0257]
    add 700 μl wash buffer, vortex, 2 min separation
  • [0258]
    discard supernatant, eventually spot 350 μl supernatant onto a filter
  • [0259]
    repeat washing procedure twice
  • [0260]
    add 120 μl elution solution, incubate 10 min. at 80° C. in a thermomixer
  • [0261]
    separate 2 min in a magnet holder, spot supernatant onto a filter
  • [0262]
    add 100 μl elution solution, resuspend and spot MGPs onto a filter
  • [0263]
    dry filters for 60 min at 75° C. in a drying oven
  • [0264]
    transfer filter into scintillation tubes, add 5 ml scintillation solution and measure in the beta-counter
  • [0265]
    5.2 Results of the Radiotracing Experiments:
  • [0266]
    5.2.1 Influence of the Magnetic Core Pigment or the Spray Dryer on the RNA Isolation
  • [0267]
    Different MGP-types, which were produced using EJ glass chemistry and various cores in the nano- or mikrorange (MMB, CERAC, etc.) on different spray dryers (Büchi or Nubilosa), were characterized with regard to their behaviour at the nucleic acid extraction from virus-negative pool material with the radiotracing method according to Example 5.1.3.2. The RNA parameter proved to be the most sensitive in the course of the studies and was therefore chosen as parameter. FIG. 11 shows BASF-CE is not suitable as core material because only small amounts of the bound RNA can be found in the eluate. The EJ/CERAC particles which were sprayed in the Büchi system at 6 bar showed the performance of the reference EJ/MMB which were sprayed in the Nubilosa system.
  • [0268]
    5.2.2 Influence of the Parameter Spray Pressure on the Isolation of DNA or RNA
  • [0269]
    Different MGP types produced with the Nubilosa system with different spraying pressures are compared according to 5.1.3.3. The reference particles are EJ/MMB MGP. The DNA and RNA binding properties are investigated. Whereas the DNA parameter shows no dependence from the spraying pressure, the RNA parameter shows significant performance differences. The performance of the EJ/CERAC particles produced with 1.5 bar spraying pressure with the Nubilosa system is lower than that of the reference. There is a lower performance in the series in dependence of the spraying pressure which varied from 1.5 to 3.4 bar (30%). Particles which are sprayed with 4.3 bar reached 90% of the performance of the particles which are sprayed at 1.5 bar (see FIG. 12).
  • [0270]
    The experiments prove the direct connection between the process parameters for the production of the MGPs and of the performance in the test. It should be noted that an increase in the spraying pressure leads to a lower test performance but, beginning from a certain spraying pressure, the particle properties are improved leading to a better performance.
  • [0271]
    5.2.3 Influence of Different MGP Production Parameters on the Isolation of RNA and DNA
  • [0272]
    These experiments should lead to the result whether a variation of the spraying pressure leads to MPGs with even better performance.
  • [0273]
    Therefor, MGPs are produced on the Nubilosa system at a pressure of 1.5 bar, 4.3 bar and 6 bar using nitrogen as spraying gas.
  • [0274]
    In order to obtain a careful drying process, inlet and outlet temperature of the spray dryer are additionally decreased. The influence of these factors on the RNA isolation is investigated according to 5.1.3.3. using EJ/MMB MGP as reference.
  • [0275]
    The results (see FIG. 13) show the surprising effect of a dramatic performance reduction which is caused by the decrease of the spraying temperature and could not be foreseen. It was possible to reach the performance of the reference quality by the increase of the spraying pressure. Concomitant with the superior suspension stability, these particles are therefore superior to the reference.
  • 6 EXAMPLE 6 Sedimentation Analytics of the Magnetic Glass Particles
  • [0276]
    6.1 Experimental Protocol
  • [0277]
    An Uvikon Spectrophotometer Model 930 produced by Kontron Instruments is used for the evaluation of the sedimentation behaviour of the magnetic glass particles. This spectrophotometer is modified to allow variable adjustment of the cuvette along a scale bar. For the measurements the cuvette is set at a position in which the measuring beam with a wavelength of 650 nm traverses the cuvette at a position which is at ⅔ of the filling height (scale position 7.5). Macro cuvettes made from polystyrene are used which have a volume of 4 ml and a path length of 1 cm. Prior to measurements, calibration is made in a one beam procedure against pure suspension medium. The MGP samples are dispersed to homogeneity in suspension medium, typically at a mass/volume ratio of 3 mg/ml, and measured immediately. The change of the extinction with time is monitored continuously at the said wavelength of 650 nm.
  • [0278]
    The physical quantity used for the comparison of the sedimentaton velocity of different samples is the half life (t1/2) in a particular suspension medium. This is the peroid of time until the extinction of the suspension in the upper third of the cuvette is half the value at the beginning of the measurement. The decline of extinction is caused by the sedimentation of the particles and the concomitant clearance of the upper third of test volume.
  • [0279]
    The above-described device further allows the installation of a magnet beneath the cuvette. Thereby the velocity of the magnetic separation can be determined.
  • [0280]
    6.2 Results of the Sedimentation Analytics
  • [0281]
    6.2.1 Experiment 1:
  • [0282]
    Several MGP-types with different cores in the range of nano- or micrometers and produced with EJ glass chemistry are investigated spectrophotometrically for their sedimentation and separation behaviour, i.e. with and without magnet under the cuvette. Mass/volume is 6 mg/ml in this case. The suspension medium is a 1:1 mixture of isopropanol and lysis buffer. The results are summarized in SEQARABIC. The particles from the CERAC type show a clear advantage in the stability of the suspension but no disadvantages for the magnetic separation. (See also FIG. 14).
  • [0283]
    6.2.2 Experiment 2:
  • [0284]
    The sedimentation behaviour of different MGPs from the type CERAC produced with EJ glass chemistry are investigated in pure isopropanol.
  • [0285]
    Important Production Parameters
  • [0286]
    EJ0100.5R-01-1.5 bar spraying pressure, 230° C. inlet temperature, Nubilosa system
  • [0287]
    EJ0108.5R-01-4.3 bar spraying pressure, 230° C. inlet temperature, Nubilosa system
  • [0288]
    EJ0096.5R-01-6.0 bar spraying pressure, 150° C. inlet temperature, Büchi system
  • [0289]
    EJ0096.5R-01 is shown in FIG. 15. These beads are largely spherical, with a highly structured surface and a size of predominantly 0.5-5 μm. The sample EJ0100.5R-01, sprayed at low pressure and high temperature, consists mainly of deformed μ-scale particles (see FIG. 16, which shows a functional equivalent to EJ0100.5R). In conclusion, particles like EJ0096.5R-01 show significant retardation of sedimentation, which is most advantageous for liquid transfers of MGP suspensions (see FIG. 14b). The results are summarized in REFMERGEFORMAT
  • 7 EXAMPLE 7 Functional Test with PCR
  • [0290]
    7.1 Heterogeneous Functional Assays: Amplification with PCR on a Perkin Elmer GeneAmp 9600® and Detection with a Biospecific Binding Test with a Chemiluminiscent-Marked Detection Probe on a Modified Elecsys1010®
  • [0291]
    7.1.1 General Considerations
  • [0292]
    Virus particles in the sample (e.g. plasma) are lysed in the presence of protease and high concentrations of chaotropic salt as well as detergent. Then, the particulate adsorbent (=MGP) is added for physico-chemical adsorption of released nucleic acids onto the glass surface, followed by magnetic separation and washing of the loaded beads, i.e. bound/free separation. Finally, dissociation of bound nucleic acids from the beads (=elution) is conducted under reversed reaction conditions relative to adsorption, i.e. with low salt buffer or even distilled water. An aliquot of eluate is then mixed with an aliquot of PCR mastermix to start amplificatin of the nucleic acids recovered in the eluate. This reaction is characterized by the equation
  • N 1 =N 0×(1+E)n, with
  • [0293]
    N0=number of molecules at the start of the polymerase chain reaction
  • [0294]
    N1=number of molecules at the end of polymerase chain reaction
  • [0295]
    E=efficiency of amplification=0≦E≦1
  • [0296]
    n=number of reaction cycles=typically 20≦n≦35
  • [0297]
    After PCR with at least one biotinylated primer, an aliquot of biotin-tagged amplificate is mixed with hybridization buffer and detection probe. After incubation, streptavidin-coated beads are added, followed by another incubation. Finally the beads are washed, signal buffer is added and the chemiluminescent signal intensity measured, which is correlated with the mass of amplified nucleic acid bound and, thus, the viral load of the plasma sample.
  • [0298]
    The expert in the field may also carry out the 1 ml protocol manually.
  • [0299]
    7.1.2 Experimental Protocol
  • [0300]
    7.1.2.1 Reagents
  • [0301]
    7.1.2.1.1 Sample Preparation
  • [0302]
    Proteinase K, liquid in glycerol/Ca-acetate, 20 mg/ml
  • [0303]
    Poly-A-RNA, 1 mg/ml, use level is a 1:1000 dilution in lysis buffer
  • [0304]
    lysis buffer, consisting of:
  • [0305]
    Tris-buffer, 50 mmol/l, pH 7.0 (1.0 & extractor protocol, respectively) or 4.0 (1.5 ml protocol)
  • [0306]
    Polydocanol, 15% (v/v) (1.0 & extractor protocol, respectively) or Triton X-100 20% (v/v) (1.5 ml protocol)
  • [0307]
    Guanidinium-isothiocyanate, 5 mol/l
  • [0308]
    1 mmol/l DTT
  • [0309]
    magnetic glass particles (MGP) either of the type EJ/BM, EJ/MMB oder EJ/CERAC suspended in isopropanol (99.8% purity) at 60 mg/ml
  • [0310]
    wash buffer consisting of
  • [0311]
    Tris-Puffer, 20 mmol/l, pH 7.5
  • [0312]
    60% Ethanol/aq (1.0 & extractor protocol, respectively) or 70% (1.5 ml protocol)
  • [0313]
    NaCl, 20 mmol/l
  • [0314]
    Eluent=double distilled water
  • [0315]
    Amplification/Mastermix:
  • [0316]
    PCR-buffer, consisting of
  • [0317]
    buffer medium
  • [0318]
    RT-PCR (Human immunodeficiency virus (HIV), Hepatitis C Virus (HCV)): 250 mmol/l Bicine/KOH pH 8.2, 557 mmol/l K-Acetat, 40% Glycerol
  • [0319]
    HBV-PCR (Hepatitis B virus (HBV)): 20 mmol/l Tris-HCl pH 8.3; 100 mmol/l KCl, 0.012% Brij 35 (=2×-concentrate)
  • [0320]
    Metal cations, MgCl2 (HBV-PCR) 3 mmol/l or MnCl2, (RT-PCR) 2.5 mmol/l (HCV) and 1.25 mmol/l (HIV)
  • [0321]
    Deoxynucleoside-triphosphate (dATP, dGTP, dCTP, dTTP, dUTP)
  • [0322]
    Analyte-specific forward-primer
  • [0323]
    Analyte-specific reverse-primer
  • [0324]
    Uracil-N-glycosidase (UNG)
  • [0325]
    DNA-polymerase (Taq- or Tth-polymerase for HBV or HIV/HCV, respectively)
  • [0326]
    H2O-demineralised (molecular biology grade) for volume adjustment ad 100 μl
  • [0327]
    Stop-Reagent for UNG After the Amplification
  • [0328]
    N-lauroylsarcosine (e.g. Roche Id.Nr. 133895), 1% w/v); 5 μl per 100 μl amplificate
  • [0329]
    7.1.2.1.2 Detection
  • [0330]
    Hybridisation buffer (e.g. Roche Id.Nr. 1930273)
  • [0331]
    chemiluminescence-marked detection probes:
  • [0332]
    HCV (Roche BMO 28.140336), working stock=8.0 mmol/l
  • [0333]
    HIV (Roche BMO 28.540948), working stock=7.8 mmol/l
  • [0334]
    HBV (Roche BMO 28.540917), working stock=9.2 mmol/l
  • [0335]
    SA-covered ECL-Beads (e.g. Roche Id.Nr. 1865943-001)
  • [0336]
    denaturation solution (e.g. Roche Id.Nr. 1930257)
  • [0337]
    ProCell (e.g. Roche Id.Nr. 1717685)
  • [0338]
    CleanCell (e.g. Roche Id.Nr. 1717642)
  • [0339]
    7.1.2.1.3 Primers and Probes
  • [0340]
    Primer:
    HIV-forward: SEQ ID NO 3
    HIV-reverse: SEQ ID NO 4
    HCV-forward: SEQ ID NO 5
    HCV-reverse: SEQ ID NO 6
    HBV-forward: SEQ ID NO 7
    HBV-reverse: SEQ ID NO 8
    Detection probes:
    HIV: SEQ ID NO 9
    HCV: SEQ ID NO 10
    HBV: SEQ ID NO 11
  • [0341]
    7.1.2.2 Reaction Conditions & Test Procedure
  • [0342]
    Sample Preparation:
  • [0343]
    1.0 ml Manual Protocol; => Used for Experiment 1 See Example 7.3.1
  • [0344]
    add 25 μl of proteinase K to 425 μl of plasma sample, vortex,
  • [0345]
    add 500 μl of lysis buffer, incubate 5 min at room temperature with mechanical agitation (1300 rpm in Eppendorf mixer),
  • [0346]
    add 50 μl of MGP suspended in isopropanol (concentration 60 mg/ml), vortex, and incubate 20 min at room temperature on a roller mixer,
  • [0347]
    magnetic separation using a Dynal magnetic separator (2 min);
  • [0348]
    remove unbound fraction through aspiration, and ad 700 μl wash buffer, vortex, separate, aspire;
  • [0349]
    repeat wash procedure another 4 times,
  • [0350]
    add 120 μl of DEPC-water, vortex, and elute 10 min at 80° C. in an Eppendorf Thermomixer at 1300 rpm with cups uncapped,
  • [0351]
    separate beads from aquous supernatant (=eluate) with Dynal magnetic separator (2 min),
  • [0352]
    freeze eluate until further use.
  • [0353]
    Automated Protocol on In-House-Built Extractor; => Used for Experiment 2 and 3 (See Example 7.3.2 and Example 7.3.3)
  • [0354]
    Basically the same procedure and reagent set as above. Additionally, an amoured RNA internal control (IC, see below) is spiked into each and every sample, the extraction process is thermostated at ca. 40° C., and volumes are adjusted to 2 ml total volume of lysis mix, i.e.
  • [0355]
    50 μl IC
  • [0356]
    50 μl proteinase K
  • [0357]
    850 μl sample
  • [0358]
    1000 μl lysis buffer
  • [0359]
    100 μl MGP suspension
  • [0360]
    2200 μl wash buffer for 5 wash steps each
  • [0361]
    125 μl eluent
  • [0362]
    1.5 ml Manual Protocol; => Used for Experiment 4 (See Example 7.3.4)
  • [0363]
    add 80 μl of proteinase K to 420 μl of plasma sample, vortex,
  • [0364]
    add 500 μl of lysis buffer, incubate 10 min at room temperature with mechanical agitation (1300 rpm in Eppendorf mixer),
  • [0365]
    add 500 μl of MGP suspended in isopropanol (concentration 6 mg/ml), vortex, and incubate 20 min at room temperature on a roller mixer,
  • [0366]
    magnetic separation using a Dynal magnetic separator (2 min);
  • [0367]
    remove unbound fraction through aspiration, and ad 700 μl wash buffer, vortex, separate, aspire;
  • [0368]
    repeat wash procedure another 4 times,
  • [0369]
    add 100 ml of DEPC-water, close the cups, vortex, and elute 15 min at 80° C. in an Eppendorf Thermomixer at 1300 rpm,
  • [0370]
    separate beads from aquous supernatant (=eluate) with Dynal magnetic separator (2 min),
  • [0371]
    freeze eluate until further use.
    amplification
    total reaction volume = 100 μl for all assays
    stock solution end concentration in PCR/μl
    HBV: eluate volume for PCR = 20 μl
    Water (PCR-Grade)   4 μl
    DNA-Mastermix 2-x 1-x/50 μl
    MgCl2  25 mM  3.0 mM/12 μl
    Primer SEQ ID NO 7   5 μM  0.2 μM/4 μl
    Primer SEQ ID NO 8   5 μM  0.4 μM/8 μl
    UNG   1 U/μl   2 U/2 μl
    HCV: eluate volume for PCR = 40 μl
    Water (PCR-Grade)   18 μl
    Bicine-Buffer RT 5x 5-x 1-x/20 μl
    MnOAc  25 mM  2.5 mM/10 μl
    DNTP-Mix (dUTP/  30 mM  0.6 mM
    DATP/dCTP/dGTP)  10 mM  0.2 mM/2 μl
    Primer SEQ ID NO 5  10 μM  300 nM/3 μl
    Primer SEQ ID NO 6  10 μM  300 nM/3 μl
    UNG   1 U/μl   2 U/2 μl
    Tth-Polymerase 5.5 U/μl   10 U/2 μl
    HIV: eluate volume for PCR = 40 μl
    Water (PCR-Grade)   20 μl
    Bicine-Buffer RT 5x 5-x 1-x/20 μl
    MnOAc  25 mM 1.25 mM/5 μl
    dNTP-Mix (dUTP/  10 mM  0.2 mM/2 μl
    dATP/dCTP/dGTP)
    Primer SEQ ID NO 3   5 μM  0.2 μM/4 μl
    Primer SEQ ID NO 4   5 μM  0.2 μM/4 μl
    UNG   1 U/μl   2 U/2 μl
    Tth-Polymerase 5.5 U/μl   15 U/3 μl
  • [0372]
    Thermocycling profiles are as follows:
    HBV: UNG-step 10 min 37° C.
    PCR 35× 30 sec 92° C.
    30 sec 55° C.
    40 sec 72° C.
    HCV: UNG-step 10 min 37° C.
    reverse transcriptase step 30 min 60° C.
    denaturation  1 min 95° C.
    PCR 10 sec 95° C.
    20 sec 60° C.
    33× 15 sec 90° C.
    20 sec 60° C.
    follow-up incubation  7 min 72° C.
    HIV: UNG-step 10 min 37° C.
    reverse transcriptase step 30 min 60° C.
    PCR 10 sec 95° C.
    10 sec 55° C.
    10 sec 72° C.
    31× 10 sec 95° C.
    10 sec 60° C.
    10 sec 72° C.
    detection on modified Elecsys 1010 ®
    volume incubation time
    reagent (HBV, HCV, HIV) (HBV, HCV, HIV)
    PCR product  10 μl
    denaturation solution  35 μl  5 min
    detection probe 120 μl 30 min
    SA-beads  35 μl 10 min
  • [0373]
    7.2 Homogeneous Functional Assays: 5′-Nuclease-Assay-Technology on a Cobas Taqman®
  • [0374]
    7.2.1 General Considerations
  • [0375]
    Lysis of viral particles as well as adsorption, purification and elution of nucleic acids extracted from the sample are conducted as described above (see Example 7.1.2). Again, amplification of target molecules is described by the equation N1=N0×(1+E)n, with
  • [0376]
    N0=number of molecules at the start of the polymerase chain reaction
  • [0377]
    N1=number of molecules at the end of polymerase chain reaction
  • [0378]
    E=efficiency of amplification=0≦E≦1
  • [0379]
    n=number of reaction cycles=typically 40≦n≦60 in this case.
  • [0380]
    With 5′-nuclease technology, however, amplification and detection reactions, respectively, are closely interwoven, and occur in solution phase, i.e. without solid phase immbilisation and corresponding washing steps (=homogeneous PCR). To this end, detection probes with 2 particular chemical modification are added to the PCR mastermix. One of these modifications is a fluorogenic reporter group (R, for instance a derivative of 6-carboxy-fluorescein) covalently atached to the backbone of the probe, the other one is a dye (for instance a polymethine-cyanine derivative) capable of absorbing the fluorescent light of the reporter and to quench it (quencher). The quencher is typically attached to the probe backbone at the 5′-end, whereas the reporter is located within the oligo sequence, spaced from the quencher by a number of nucleotide building blocks. These probes bind to the target nucleic acid (sense or anti-sense strand) close to the 3′-end of a primer (reverse or forward). As soon as primer has annealed to the target and DNA-polymerse gets bound to the primer:target hybrid, elongation starts. Due to the 5′-nuclease activity of the enzyme, simultaneously with copy strand synthesis the probe is cleaved as soon as the polymerase reaches the probe binding site, reporter and quencher get separated and the fluorescent signal becomes measurable. This process is repeated with every cycle, and more and more fluorescent reporter is accumulated in solution until reagent depletion at the end of the reaction. So, in a signal-over-time plot, sigmoidal growth curves are generated. The bigger No, the earlier the signal curve rises above noise level.
  • [0381]
    The point on the time axis, where the fluorescent signal can be significantly distinguished from background singal is called threshold cycle (ct). ct is a measure of assay sensitivity: the smaller the ct-value, the more sensitive is the assay. ct values can be calculated by means of different mathematical operations, for example cut-off approaches (average background signal intensity multiplied by a constant factor yields a cut-off signal intensity to discern negative from positive) or approaches where the location of the maximum of the first differentiation of the signal-over-time curve (i.e. the steepness profile), or the location of the maximum of the second differentiation on the time axis, are computed and defined as ct.
  • [0382]
    This amplification/detection technology allows for real-time monitoring of PCR and for closed-tube processing as well, i.e. in contrast to standard procedures, the PCR tubes remains closed after pipetting eluate and mastermix which effectively reduces contamination risks.
  • [0383]
    Moreover, if one uses primer and probe sets for different viral species (and, thus, different analyte molecules and target sequences) combined in one mastermix, it is possible to perform multiple amplification/detection reactions simultaneously, depending on the viral load of the individual under inspection. This is the basis for so-called multiplex assays.
  • [0384]
    What is more, due to the generic nature of MGP based sample preparation, all nucleic acids present in the sample are extracted by way of physico-chemical adsorption to the particle surface, independent of sequence characteristics. This will also include human DNA (hDNA), released from disrupted blood cells, e.g. leucocytes, the titer of which may differ widely according to the physiological or pathological status of the individual blood sample donor. For instance, in cases of autoimmune diseases like SLE, hDNA levels may be elevated substantially. So, hDNA and one or more pathogenic nucleic acids present in a given sample constitute a mixture of various nucleic acids of different sequence specificities which are co-extracted. They constitute a matrix of polynucleotides that are extracted without discrimination, followed by sequence-specific amplification and detection of target nucleic acids via interaction with specific primers and probes under appropriate reaction conditions.
  • [0385]
    Given a pathological level of 4000 ng/ml hDNA in plasma in case of SLE, and a low viral load of 50 copies of viral genomic RNA per ml of plasma., and 10000 nucleotides per genomic RNA. With ca. 325 dalton (1 dalton=1.66×1024 g) per nucleotide, 50 copies or 500000 nucleotides of target RNA make ca. 2.7×10−7 ng per ml of plasma, resulting in a relative abundance of target: non-target nucleic acid of approximately 1:10+12!
  • [0386]
    hat is more, in order to monitor the whole process, an artificial nucleic acid construct may be added to the samples, preferably packaged (armored) in a modified virus particle, which is co-extracted and co-amplified with the natural target. This internal control (IC) features a unique probe binding region for an IC detection probe, which differs from target-specific probes by a different reporter group with distinguishable emission characteristics. Thus, IC signal can be discerned from target signal, and as the IC is known to be present in the sample, it functions as a monitoring agent. So, multi-labeling expands the utility of multiplex assay technology. The internal control (IC) is described in WO98/00547.
  • [0387]
    7.2.2 Experimental Protocol
  • [0388]
    7.2.2.1 Reagents
  • [0389]
    7.2.2.1.1 Sample Preparation
  • [0390]
    See above (Example 7.1.2=>automated protocol on in-house-built extractor)
  • [0391]
    7.2.2.1.2 5′-Nuclease-Assay, Reactants & Reaction Conditions
    Concentration Final
    Addition concentration (μl/
    Reagent of stock solution in the test reaction)
    Mn(OAc)2 pH 6.5   50 mM  3 mM 5.75
    dNTPs 2.50
    (dG, dA, dC)  100 mM 300 μM
    (dT)  50 μM
    (dU) 500 μM
    DEPC-H20 9.50
    Glycerol 80% 5,64% in total 3.50
    2.8% added here*
    K Oac pH 7.5   2 M 100 mM 5.00
    Tricine pH 8.3   1 M  50 mM 5.00
    DMSO 80% 5.0% 6.25
    Primer SEQ ID NO 12   50 μM 150 nM 0.30
    Primer SEQ ID NO 13   50 μM 150 nM 0.30
    Primer SEQ ID NO 14   50 μM 400 nM 0.80
    Primer SEQ ID NO 15   50 μM 150 nM 0.30
    Primer SEQ ID NO 16   50 μM 400 nM 0.80
    Primer SEQ ID NO 17   50 μM 150 nM 0.30
    Primer SEQ ID NO 18   50 μM 150 nM 0.30
    Probe SEQ ID NO 19   50 μM 100 nM 0.20
    Probe SEQ ID NO 20   50 μM 100 nM 0.20
    Probe SEQ ID NO 21   50 μM 100 nM 0.20
    Probe SEQ ID NO 22   50 μM 100 nM 0.20
    UNG  2.4 U/μl  10 U 4.20
    ZO5   10 U/μl  40 U 4.00
    Aptamer 50.4 μM 200 nM 0.40
    SEQ ID NO 23
  • [0392]
    The common multiplex thermocycling profile for all test parameters is as follows:
    UNG-step 45° C. - 10 min
    denaturation 94° C. - 30 sec
    reverse transcription 58° C. - 30 min
    PCR 95° C. - 20 sec/59° C. - 50 sec  5×
    91° C. - 15 sec/52° C. - 50 sec 55×
    7.2.2.1.3 Sequences
    Primer:
    HIV-forward SEQ ID NO 12
    HIV-forward SEQ ID NO 13
    HIV-reverse SEQ ID NO 14
    HCV-forward SEQ ID NO 15
    HCV-reverse SEQ ID NO 16
    HBV-forward SEQ ID NO 17
    HBV-reverse SEQ ID NO 18
    Probes:
    HIV SEQ ID NO 19
    HCV SEQ ID NO 20
    HBV SEQ ID NO 21
    IC SEQ ID NO 22
    Aptamer SEQ ID NO 23
  • [0393]
    Terminology of the Chemical Derivatisations:
  • [0394]
    Some of the oligonucleotides are derivatised with Cy5 which is a Pentamethine-di-indocarbocyanine coupled to alkylphosphatidyl-linker (Pharmacia Biotech Cy5-N-ethyl-phosphoramidite) and which functions as quencher (Q); λEX=630 nm, λEM=665 nm. Some of the oligonucleotides are derivatised with FAM which is 6-carboxy-fluorescein coupled to a 2-(amino-cyclohexyl-)propane-1,3-diol-linker (Biogenex CX-FAM-phosphoramidite) and which functions as s reporter for targets (R); λEX=485 nm, λEM=515 nm. Some of the oligonucleotides are derivatised with which is hexachloro-6-carboxy-fluorescein coupled to a 2-(amino-cyclohexyl-)propane-1,3-diol-linker (Biogenex CX-HEX-phosphoramidite) and which functions as reporter for the internal control (R); λEX=530 nm, λEM=585 nm
  • [0395]
    7.2.2.1.4 Reaction Conditions
  • [0396]
    The reaction conditions are described in the results of the experiments (see Example 7.3).
  • [0397]
    7.2.2.1.5 Other Materials
  • [0398]
    virus-negative plasma (O-Matrix, single plasma or Plasma-Pool), e.g. citrate-plasma or EDTA-plasma
  • [0399]
    virus-positive plasma or virus-containing culture supernatants, which can be used to adjust certain virus titers by mixing with O-matrix in appropriate ratios.
  • [0400]
    alternatively: in vitro-transcripts with the target-sequences to be investigated
  • [0401]
    human placental DNA (a. genomic, Sigma cat. no. D4642; b. fragmented, Sigma cat.no. D3287), added to the samples to simulate pathological conditions which lead to the enhanced release of intracellular substances and thus to enhanced levels of DNA/RNA in the blood, e.g. auto immune disease as SLE or hemolysis.
  • [0402]
    7.3 Results of the Functional Tests
  • [0403]
    7.3.1 Experiment 1
  • [0404]
    Different MGP-samples of the type EJ/CERAC are investigated with the methods described in 7.1. The test variable was the spraying pressure:
  • [0405]
    EJ0100.5R-1.5 bar
  • [0406]
    EJ0106.5R-2.5 bar
  • [0407]
    EJ0107.5R-3.5 bar
  • [0408]
    EJ0108.5R-4.3 bar
  • [0409]
    It could be demonstrated that with increasing spraying pressure, i.e. shift towards smaller average bead diameter, the unspecific binding (USB) decreases whereas the highly specific signal generation is unchanged which results in elevated signal-to-noise ratios
  • [0410]
    The virus titer adjustment 0.5×GG is approximately 100 sgu/ml (sgu=signal generating units) in the case of HBV, and approximately 150 sgu/ml in the case of HIV. The data are summarized in REFMERGEFORMATTable 7.
  • [0411]
    7.3.2 Experiment 2:
  • [0412]
    Two variants each of the type EJ/MMB and EJ/CERAC, respectively, are functionally compared using the method as described in Example 7.2 in conjunction with multipool (MP) and individual (PL) plasma samples. EJ0047.2R (MMB) and EJ0100.5R (CERAC) are sprayed under standard conditions, i.e. 1.5 bar, 230° C. inlet temperature and approximately 110° C. outlet temperature. EJ0102.2R (MMB) and EJ0108.5R (CERAC) are development variants with regard to the spraying conditions (inlet temperature decreased to 200° C. [MMB]), and spraying pressure increased to 4.3 bar [CERAC]). The results are summarized in REFMERGEFORMATTable 8 and show the potential of the CERAC nano-core particles to obtain higher sensitivity as exemplified by the earlier threshold cycle and/or the larger signal differences (saturation signal minus noise level, S−N).
  • [0413]
    7.3.3 Experiment 3
  • [0414]
    Several MGP preparations of the type EJ/MMB and EJ/CERAC (see REFMERGEFORMATTable 9) are functionally compared using the method as described in Example 7.2. The results are summarized in REFMERGEFORMATTable 10 and show the potential of the of the EJ0096.5R-01-type particles to obtain higher sensitivity as exemplified by the earlier threshold cycle or higher hit rate irrespective of the particular assay.
  • [0415]
    7.3.4 Experiment 4
  • [0416]
    Several MGP preparations of the type EJ/MMB and EJ/CERAC are functionally compared using the method as described in Example 7.1 wherein the adsorption incubation is conducted with or without shaking. Thereby, the sedimentation velocity becomes decisive, i.e. the higher the sedimentation velocity the higher the number of particles which are removed from the interaction with the analyte in the forming precipitate. In contrast, if the sedimentation velocity is smaller then time span is longer in which analyte molecules from the liquid may adsorb to the surface of the adsorbent. The results are summarized in REFMERGEFORMATTable 11 (EJ0047.2R was used as a reference) show that without mechanical agitation the loss of performance is highest (>40%) with the MMB-type (differs considerably from the characteristics of this invention), and least, or practically absent, with the CERAC-type (most close to the characteristics of the invention within this series).
  • [0417]
    To be able to assess the functionality of the different types of MGP thoroughly, a performance index was calculated for each type in the following way:
  • [0418]
    the eluate of several batches for each type of MGP and each titer level, respectively, are pooled into 1 eluate pool
  • [0419]
    out of each eluate pool, 3 amplifications were performed for each assay (HIV, HCV, HBV)
  • [0420]
    each amplificate was measured in singlicate on the modified Elecsys 1010®
  • [0421]
    signals are averaged for each type of MGP, assay, and titer level
  • [0422]
    signal-to-noise (S/N) factors are calculated for each type of MGP and assay, respectively, i.e. low titer (weakly positive) over O-matrix (negative), and elevated titer over O-matrix
  • [0423]
    S/N factors are normalized to the reference type of MGP at the time
  • [0424]
    calculation of the sum of normalized S/N factors for each type of MGP, cumulated over all assays and titer levels; S/N factors for the low titer range are weighted 2-fold relative to those for the high titer level so as to pronounce sensitivity aspects
  • [0425]
    the resulting sum is assigned performance index for each type of MGP investigated
  • [0426]
    7.3.5 Experiment 5
  • [0427]
    Different types of MGP were used to extract HCV in vitro transcripts which were diluted to various titer levels in diluent containing 10 mmol/l Tris, pH 80, 1 mM EDTA, 20 μg/ml poly-A-RNA and 0.05% NaN3, and spiked into pooled plasma. Human background DNA (hBG-DNA) was spiked at 0 or 4000 ng/ml into the sample, respectively. Surprisingly, it is possible to reduce interference by hBG-DNA by way of bead selection, as demonstrated by the data presented in Table 12.
  • [0428]
    7.3.6 Experiment 6
  • [0429]
    Various types of MGP were used to process HCV positive plasma samples. Human background DNA (hBG-DNA) was spiked at 0 or 4000 ng/ml into the sample, respectively. Again, a significant impact of bead size and bead geometry is being observed. MGP preparation EJ0096.5R, which represents the MGP quality according to the present invention the most, shows very clearcut advantages both in terms of minimal shift of ct values, and reducing loss of specific signal generation (S—N), as indicated by the data presented in Table 13.
    TABLE 1
    Code (see
    Example
    Company Abbreviation Description 1.4)
    No pigment 0
    Merck BM Merck Black Mica: 1
    Darmstadt, Small plates of mica (support), diameter = 10-60 μm,
    Germany thickness approximately 1-2 μm, Fe3O4-ashlars and
    TiO2-grains are attached; typically, several pigment
    units are aggregated (Merck Iriodin ®)
    Merck MMB Merck Microna Matte Black 2
    Darmstadt, Small plates of mica (support), diameter = 3-15 μm,
    Germany thickness approximately 1-2 μm, Fe3O4-ashlars are
    attached; typically, several pigment units are
    aggregated
    BASF BASFFA BASF iron oxide FA28-41: 4
    Ludwigshafen, Needles in the nanoscale range made from γ-Fe2O3,
    Germany length approximately 200-400 nm, very high tendency
    to aggregate to particles in the μ-scale because of the
    form and large highly structured surface
    Cerac, CERAC CERAC γ-Fe2O3 (Catalogue number I-2012, 5
    Milwaukee, distributed by Chemco Chemieprodukte GmbH in
    WI, USA Germany) γ-Fe2O3-microbeads in the nanoscale
    (pure ferric oxide), diameter = 23 nm, non-aggregating
    Strem STREM STREM Fe3O4 > 95% (Catalogue-No.: 93-2616) 7
    Chemicals, Microbeads in the nanoscale range made from
    Newburyport, precipitated Fe3O4, diameter = 200-500 nm, strong
    MA, tendency to aggregate to particles in the μ-range
    USA
    BASF, BASFCE Beads in the μ-scale made from metallic iron
    Ludwigshafen, BASF Carbonyl iron powder HQ: 10% < 1.04 μm, 8
    Germany 50% < 1.91 μm, 90% < 3.71 μm
    BASF Carbonyl iron powder SU: 10% < 0.43 μm, 9
    50% < 0.93 μm, 90% < 1.81 μm
  • [0430]
    [0430]
    TABLE 2
    temperature
    Water Alcohol inlet Temperature temperature temperature treatment
    (ml/ (ml/ spray tempe- in air in N2 in O2 in bottle
    MI 100 ml 100 ml Ageing pressure rature before FA after FA after FA under air
    Code TEOS TEOS) Alcohol TEOS) (h) (bar) Nozzle (° C.) (° C.) (° C.) (° C.) (° C.)
    0005 1750 13.33 EtOH 50 1 1.5 A 230 250 675 300 300
    0009 1750 13.33 EtOH 50 1 1.5 A 230 250 675 300 300
    temperature temperature
    Water Alcohol inlet (post) treatment
    (ml/ (ml/ Pigment Spray tempe- Temperature treatment in bottle
    MI 100 ml 100 ml Ageing (g/100 g pressure rature treatment temperature in air under air
    Code TEOS TEOS) Alcohol TEOS) (h) Oxid) (bar) Nozzle (° C.) in air (° C.) in N2 (° C.) (° C.) (° C.)
    0045 1400 13.33 EtOH 50 0 1.5 A 230 200 675 300 200
    0046 250 13.33 EtOH 50 0 5 Büchi 150 200 675 300 200
    0047 1300 14.13 EtOH 137 0 1.5 A 230 250 750 200 200
    0096 150 14.1 EtOH 141 0 230 6 Büchi 150 200 750 200 200
    0097 150 14.1 EtOH 141 0 191 6 Büchi 150 200 750 200 200
    0098 210 14.1 EtOH 141 0 230 6 Büchi 150 200 690 200 200
    0099 210 14.1 EtOH 141 0 230 6 Büchi 150 200 710 200 200
    0100 840 14.1 EtOH 141 0 230 1.5 A 230 200 750 200 200
    0101 840 14.1 EtOH 141 0 191 1.5 A 230 200 750 200 200
    0102 1300 14.1 EtOH 141 0 230 1.5 A 230 200 750 200 200
    0106 840 14.1 EtOH 141 0 230 2.5 A 230 200 750 200 200
    0107 3.5 A 230 200 750 200 200
    0108 4.3 A 230 200 750 200 200
    0109 8000 14.1 EtOH 141 0 230 1.5 A 210 200 750 200 200
    0111 1300 14.1 EtOH 141 0 230 1.5 A 210 200 750 200 200
    0119 1200 14.6 EtOH 130 0 230 1.5 A 210 200 750 200 200
    0124 6 Büchi 150 200 750 200 200
    0128 7830 14.1 EtOH 141 0 230 1.5 A 230 200 750 200 200
    0129 1.5 A 200 200 750 200 200
    0130 4.3 A 200 200 750 200 200
    0131 6 A 200 200 750 200 200
  • [0431]
    [0431]
    TABLE 3
    Outlet
    temperature Density Extinction Extinction Magnetic force
    Sample (° C.) (g/cm3) (280 nm) (400 nm) Iron solubility (mg/l) (g/250 mg)
    RN0005.2E-01 116 3.420 0.037 0.027 <determination limit 16.04
    RN0009.1E-01 119 3.155 0.106 0.093 <determination limit 9.50
    EJ0028.2R-01 118 3.602 0.019 0.024 <determination limit 12.25
    RN0045.4R-01 130 3.647 0.032 0.034 <determination limit 9.23
    RN0046.7R-01 98 3.866 0.013 0.013 <determination limit 13.12
    RN0045.5R-01 120 3.802 0.019 0.017 0.047 9.52
    EJ0047.2R-01 118 3.659 0.022 0.018 <determination limit 12.00
    EJ0096.5R-01 96 4.203 0.001 0.000 <determination limit 13.53
    EJ0096.7R-01 96 4.328 0.000 0.001 <determination limit 18.36
    EJ0097.4R-01 96 3.859 0.001 0.000 <determination limit 15.17
    EJ0098.8R-01 95 5.588 0.065 0.055 <determination limit 15.76
    EJ0099.9R-01 95 5.538 0.103 0.101 0.016 14.13
    EJ0100.7R-01 122 4.196 0.027 0.003 <determination limit 16.29
    EJ0100.5R-01 126 4.069 0.012 0.012 <determination limit 12.42
    EJ0101.4R-01 119 4.079 0.083 0.084 0.015 11.67
    EJ0102.2R-01 128 3.668 0.009 0.009 <determination limit 13.36
    EJ0047.2R-07 118 3.612 0.024 0.022 0.159 11.26
    EJ0100.5R-02 127 4.107 0.029 0.023 0.054 14.51
    EJ0100.5R-03 127 4.173 0.063 0.045 0.126 10.44
    EJ0106.5R-01 127 4.191 0.004 0.004 0.025 10.09
    EJ0107.5R-01 127 4.235 0.016 0.009 0.037 10.25
    EJ0108.5R-01 127 4.233 0.026 0.026 0.039 11.56
    EJ0109.2R-01 110 3.700 0.046 0.043 0.028 11.23
    EJ0111.5R-01 117 3.612 0.071 0.036 0.166 16.48
    EJ0111.5R-02 121 4.082 0.065 0.040 0.161 15.95
    EJ0124.5R-01 3.992 0.001 0.001 0.051 17.57
    EJ0128.5R-01 125 4.078 0.023 0.028 8.97
    EJ0129.5R-01 92 4.013 0.044 0.043 9.64
    EJ0130.5R-01 87 4.022 0.031 0.036 11.70
    EJ0131.5R-01 89 3.908 0.033 0.037 11.73
    EP0119.5R-01 127 4.110 0.003 0.003 0.079 17.15
  • [0432]
    [0432]
    TABLE 4
    BET surface
    Lot [m2/g] Remarks
    EJ0096.5R-01 26.85 Cerac, small spray dryer, 6 bar
    EJ0100.5R-01 8.95 Cerac, big spray dryer, 1, 5 bar
    EJ0101.4R-01 8.47 BASF FA, big spray dryer, 1, 5 bar
    RN0005.2E-01 3.54 MERCK MMB, big spray dryer, 1, 5 bar
    RN0009.1E-01 3.25 MERCK BM, big spray dryer, 1, 5 bar
    EJ0099.9R 0.74 BASF CE-SU, big spray dryer, 6 bar
    EJ0100.7R 8.53 STREM, big spray dryer, 1, 5 bar
  • [0433]
    [0433]
    SEQARABICTABLE 5
    t1/2 t1/2
    Size of the sedimen- separa-
    isolated tation tion
    MGP-type cores Constitution Form [min] [sec]
    MMB   3-15 μm Aggregated Plates 3.9 14
    CERAC    23 nm Singular Beads >>10 16
    STREM 300-500 nm Aggregated Beads 4.8 10
    BASF-FA 200-300 nm Aggregated Needles 4.6 12
  • [0434]
    [0434]
    SEQARABICTABLE 6
    t0,9 (90%-Wert) t1/2 (50%-Wert)
    MGP-sample sedimentation [min] sedimentation [min]
    EJ0100.5R-01 0.35 2.78
    EJ0108.5R-01 0.62 7.58
    EJ0096.5R-01 1.39 19.85
  • [0435]
    [0435]
    TABLE 7
    USB [counts] - 0-matrix SB [counts] - 0.5xGG
    MGP HIV HBV HIV HBV
    EJ0100.5R-01 4997 2277 20380 80292
    EJ0100.5R-02 6100 3590 22036 53417
    EJ0100.5R-03 4856 2270 19791 65109
    EJ0106.5R-01 4427 2263 19030 75128
    EJ0107.5R-01 3940 2261 19255 68285
    EJ0108.5R-01 3429 2217 20048 80426
  • [0436]
    [0436]
    SEQARABICTABLE 8
    HBV HCV
    Sample MGP lot# Ct S-N ct S-N
    MP2 EJ0047.2 40.49 19.88 41.68 12.6
    MP2 EJ100.5 40.96 21.80 43.08 12.74
    MP2 EJ0102.2 41.93 21.39 44.74 10.10
    MP2 EJ0108.5 40.09 22.01 47.36 10.61
    P1. ½ EJ0047.2 41.03 18.14
    P1. ½ EJ100.5 40.12 19.31
    P1. ½ EJ0102.2 41.81 22.80 44.69 14.30
    P1. ½ EJ0108.5 39.93 21.77 43.27 12.58
    P1. ⅘ EJ0047.2 46.33 12.83 39.54 18.30
    P1. ⅘ EJ100.5 40.82 15.12 38.24 21.48
    P1. ⅘ EJ0102.2 42.49 19.65 39.24 19.05
    P1. ⅘ EJ0108.5 47.33 9.51 39.47 17.81
    P1. {fraction (6/8)} EJ0047.2 41.08 20.83 49.44 9.46
    P1. {fraction (6/8)} EJ100.5 40.68 23.16 48.98 10.31
    P1. {fraction (6/8)} EJ0102.2 40.50 22.49 54.50 6.50
    P1. {fraction (6/8)} EJ0108.5 38.79 18.60 50.41 8.61
    P1. {fraction (6/8)} EJ0047.2 44.16 17.26 41.40 10.59
    P1. {fraction (6/8)} EJ100.5 42.97 18.59 41.01 11.33
    P1. {fraction (6/8)} EJ0102.2 44.49 12.41 42.18 11.32
    P1. {fraction (6/8)} EJ0108.5 41.24 18.73 41.46 10.80
    Counting statistics: Which type scores better ?
    total: best type per
    “standard samples” “development variants” sample * assay
    MMB:CERAC MMB:CERAC MMB:CERAC
    Ct S-N Ct S-N Ct S-N
    2:7 0:9 3:7 5:5 1:9 3:7
  • [0437]
    [0437]
    TABLE 9
    MGP Description
    EJ0096.5R 6.0 bar spraying pressure, 150° C. inlet temperature, Büchi-
    laboratory equipment. This preparation approximates
    the characteristics according to the
    invention very well, see FIG. 15.
    EJ0111.5R 1.5 bar spraying pressure, 230° C. inlet
    temperature, Nubilosa-laboratory equipment
    EP0119.5R 1.5 bar spraying pressure, 230° C. inlet
    temperature, Nubilosa-laboratory
    equipment, changed glass composition without
    Zn-acetate, K-methanolate
    substituted by K-acetate, with identical stoichometry
    EJ108.5R 4.3 bar spraying pressure, 230° C. inlet
    temperature, Nubilosa-laboratory equipment
  • [0438]
    [0438]
    SEQARABICTABLE 10
    Pa- Titer MGP lot MGP lot MGP lot MGP lot
    ram- [sgu/ EJ0111.5R-02 EP0119.5R EJ0096.5R EJ0108.5R
    eter ml] AVG AVG AVG AVG
    ct-values (HBV)
    ct-calculation via maximum of first differentiation
    HBV NK
    100 46.1 44.1(6/6) 43.4 44.2
    40 47.6 46.5(7/8) 45.5(7/8) 46.7(6/7)
    20 50.6(6/8) 46.1(6/8) 46.8(7/8) 50.0(4/8)
    10 47.6(5/8) 50.1(5/8) 46.8(4/8) 47.7(3/7)
    5 51.2(3/8) 48.6(4/8) 47.8(5/8) 49.7(4/7)
    hit 75% 74% 77.5% 67.5%
    rate
    ct-calculation via cut-off formula
    HBV NK
    100 44.2 41.7(6/6) 41.5 41.9
    40 45.5 44.1(7/8) 42.7(6/8) 45.0(6/7)
    20 47.7(6/8) 43.5(6/8) 44.3(7/8) 47.2(6/8)
    10 45.5(5/8) 47.7(5/8) 44.4(6/8) 45.6(3/8)
    5 49.3(3/8) 45.7(4/8) 45.4(4/8) 46.6(4/7)
    hit 75% 74% 77.5% 71%
    rate
    Pa- Titer MGP lot MGP lot MGP lot MGP lot
    ram- [IU/ EJ0111.5R-02 EP0119.5R EJ0096.5R EJ0108.5R
    eter ml] AVG AVG AVG AVG
    ct-values (HCV)
    ct-calculation via maximum of first differentiation
    HCV NK
    400 42.7 42.5(7/8) 41.8 42.6(7/7)
    80 44.4(7/8) 44.1(6/6) 44.2(7/8) 44.2(7/7)
    60 45.1 44.7(7/7) 44.4(6/7) 45.1(6/6)
    40 46.2(7/8) 45.8(7/7) 43.9(5/7) 44.5(7/7)
    20 46.5(5/8) 46.3(7/7) 45.0(6/8) 47.4(4/8)
    hit 90% 97% 84% 88.5%
    rate
    ct-calculation via cut-off formula
    HCV NK
    400 41.8 41.6(7/8) 40.9 41.7(7/7)
    80 44.6(7/7) 44.0(6/6) 43.4(7/8) 44.5(7/7)
    60 44.7 45.3(7/7) 45.1(6/7) 44.3(5/6)
    40 47.9(7/8) 46.1(7/7) 44.7(4/7) 47.5(7/7)
    20 49.6(5/8) 47.2(7/7) 45.3(5/8) 47.4(4/8)
    hit 90% 97% 79% 86%
    rate
    ct-values (HIV)
    ct-calculation via maximum of first differentiation
    Pa- Titer MGP lot MGP lot MGP lot MGP lot
    ram- Titer EJ0111.5R EP0119.5R EJ0096.5R EJ0108.5R
    eter [sgu/ml] AVG AVG AVG AVG
    HIV NK
    30 43.2(6/8) 46.9(3/8) 43.9(3/7) 41.9(1/7)
    60 42.7(4/7) 42.6(4/7) 42.8(7/7) 42.4(2/7)
    150 42.4(5/7) 42.0 41.7(6/6) 42.6(6/8)
    300 41.9(5/5) 41.0(5/5) 41.5(6/6) 41.3(6/6)
    600 41.6(7/7) 40.8(6/6) 40.8(5/5) 41.5
    hit rate 79% 76% 87% 64.0%
    ct-calculation via cut-off formula
    Pa- Titer MGP lot MGP lot MGP lot MGP lot
    ram- Titer EJ0111.5R EP0119.5R EJ0096.5R EJ0108.5R
    eter [IU/ml] AVG AVG AVG AVG
    HIV NK
    30 44.3(1/8) 47.3(2/8) 46.8(1/7) —(0/7)
    60 50.3(3/7) 54.1(1/7) 51.4(4/7) —(0/7)
    150 47.4(5/7) 43.7(5/8) 45.5(5/7) 52.2(2/8)
    300 42.2(5/5) 44.8(5/5) 41.6(4/6) 46.5(6/6)
    600 41.2(7/7) 41.3(5/6) 41.1(5/5) 42.5
    hit rate 62% 53% 59% 44%
  • [0439]
    [0439]
    SEQARABICTABLE 11
    ratio of
    Performance performance
    MGP type HIV I HCV HBV index indices
    60 sgu/ 600 sgu/ 480 sgu/ 4800 40 sgu/ 400 sgu/ Cumulated/ Without/with
    neg neg neg sgu/neg neg neg weighted/ shaking
    normalized
    EJ0047.2R 100 100 100 100 100 100 900
    (reference)
    EJ0096.5R 55 34 44 74 146 112 709
    EJ0096.7R 47 47 18 8 116 107 523
    EJ0097.4R 32 54 29 47 169 132 692
    RN0045.5R 85 64 76 63 7 84 545
    RN0046.7R 109 80 43 40 121 135 803
    RN0045.4R 48 93 112 128 136 96 908
    EJ0047.2R 24 24 52 49 93 122 534 0.59
    (reference)
    EJ0096.5R 28 23 47 88 102 154 619 0.87
    EJ0096.7R 34 23 38 14 99 70 449 0.86
    EJ0097.4R 33 36 20 18 101 94 456 0.66
    RN0045.5R 53 74 60 25 107 72 610 1.12
    RN0046.7R 141 90 16 8 32 40 518 0.64
    RN0045.4R 55 113 25 146 78 101 675 0.74
  • [0440]
    [0440]
    TABLE 12
    HCV-Titer
    particles (IVT-RNA) ΔhDNA Δct ΔS-N
    RN0009.1E = big platelets 1000 cp/PCR 4000 ng/ml +2 −6.1
    (3-60 μm) EJ0100.5R = deformed balls (ca. 1-12 μ-range) EJ0108.5R = deformed balls* (ca. 1-4 μ-range) −1  ±0 −3.2  −1.2
  • [0441]
    [0441]
    TABLE 13
    HCV-Titer
    (extracted
    particles genomic
    (EJ/CERAC throughout) RNA) ΔhDNA Δct ΔS-N
    EJ0096.5R = design goal* 200 IU/ 4000 ng/ml −0.95 −8.5
    ml sample
    EJ0127.5R = standard EJ0129.5R = p-variant EJ0130.5R = p-variant EJ0131.5R = p-variant −3.08 −2.30 −2.62 −2.46 −13.89 −12.62 −10.57 −12.41
  • [0442]
    [0442]
  • 1 23 1 23 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 1 tcagggcaaa actcagctca ccg 23 2 23 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 2 gatgagttcg tgtccgtaca act 23 3 30 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer (HIV forward) 3 agttggagga catcaagcag ccatgcaaat 30 4 27 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer (HIV reverse) 4 tgctatgtca gttccccttg gttctct 27 5 24 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer (HCV forward) 5 gcagaaagcg tctagccatg gcgt 24 6 24 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer (HCV reverse) 6 ctcgcaagca ccctatcagg cagt 24 7 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer (HBV forward) 7 ggagtgtgga ttcgcact 18 8 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer (HBV reverse) 8 tgagatcttc tgcgacgc 18 9 20 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe (HIV) 9 atcaatgagg aagctgcaga 20 10 21 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe (HCV) 10 gtcgtgcagc ctccaggacc c 21 11 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe (HCV) 11 agaccaccaa atgcccct 18 12 29 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer (HIV) 12 agtgggggga catcaagcag ccatgcaaa 29 13 29 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer (HIV) 13 agtgggggga caccaggcag caatgcaaa 29 14 30 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer (HIV reverse) 14 ggtactagta gttcctgcta tgtcacttcc 30 15 26 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer (HCV forward) 15 gcagaaagcg tctagccatg gcgtta 26 16 28 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide (HCV reverse) 16 gcaagcaccc tatcaggcag taccacaa 28 17 28 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer (HBV forward) 17 agaagtcaga aggcaaaaan gagagtaa 28 18 27 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer (HBV reverse) 18 cacctctgcc taatcatctc ttgttca 27 19 32 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe (HIV) 19 tctgcagctn tcctcattga tggtatcttt ta 32 20 32 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe (HCV) 20 cggtgtactc accgnttccg cagaccacta tg 32 21 33 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 21 ccaagctgtg ccttggntgg ctttggggca tgg 33 22 31 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 22 tggactcagt cctntggtca tctcaccttc t 31 23 46 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide (Aptamer) 23 cgatcatctc agaacattct tagcgttttg ttcttgtgta tgatcg 46
Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US2885366 *Jun 28, 1956May 5, 1959Du PontProduct comprising a skin of dense, hydrated amorphous silica bound upon a core of another solid material and process of making same
US2913419 *Apr 18, 1956Nov 17, 1959Du PontChemical process and composition
US3926659 *Mar 15, 1974Dec 16, 1975Merck Patent GmbhIron-containing mica flake pigments
US3945862 *Nov 15, 1974Mar 23, 1976Merck & Co., Inc.Coated ferrous substrates comprising an amorphous magnesia-silica complex
US4082905 *May 16, 1977Apr 4, 1978Bayer AktiengesellschaftProduction of iron oxide pigments with improved resistance to oxidation
US4124385 *Dec 2, 1976Nov 7, 1978Xerox CorporationMagnetic glass carrier materials
US4124735 *Dec 2, 1976Nov 7, 1978Xerox CorporationMagnetic glass carrier materials
US4126437 *Dec 2, 1976Nov 21, 1978Xerox CorporationMagnetic glass carrier materials
US4233169 *Apr 13, 1979Nov 11, 1980Corning Glass WorksPorous magnetic glass structure
US4280918 *Mar 10, 1980Jul 28, 1981International Business Machines CorporationMagnetic particle dispersions
US4297337 *Apr 13, 1979Oct 27, 1981Corning Glass WorksSolid-phase immunoassays using magnetic glass
US4309459 *Nov 25, 1980Jan 5, 1982Tdk Electronics Co., Ltd.Process for producing SiO2 coated iron oxide powder for use in the preparation of acicular magnetic iron or iron oxide powder
US4336310 *Nov 19, 1980Jun 22, 1982Tdk Electronics Co., Ltd.Magnetic recording medium and preparation thereof
US4360441 *Jun 25, 1981Nov 23, 1982Corning Glass WorksGlass-encapsulated magnetic materials and methods for making them
US4395271 *May 9, 1980Jul 26, 1983Corning Glass WorksMethod for making porous magnetic glass and crystal-containing structures
US4554088 *May 12, 1983Nov 19, 1985Advanced Magnetics Inc.Magnetic particles for use in separations
US4564537 *Jan 18, 1985Jan 14, 1986The British Petroleum Company P.L.C.Process for depositing a silica coating on a metal surface
US4628037 *Jun 13, 1985Dec 9, 1986Advanced Magnetics, Inc.Binding assays employing magnetic particles
US4672040 *Jun 28, 1985Jun 9, 1987Advanced Magnetics, Inc.Magnetic particles for use in separations
US4683195 *Feb 7, 1986Jul 28, 1987Cetus CorporationProcess for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683202 *Oct 25, 1985Nov 27, 1990Cetus CorpTitle not available
US4695392 *Jun 13, 1985Sep 22, 1987Advanced Magnetics Inc.Magnetic particles for use in separations
US4695393 *Jun 13, 1985Sep 22, 1987Advanced Magnetics Inc.Magnetic particles for use in separations
US4698302 *Jun 13, 1985Oct 6, 1987Advanced Magnetics, Inc.Enzymatic reactions using magnetic particles
US4699717 *Feb 14, 1986Oct 13, 1987Detlev RiesnerChromatographic process for the separation of nucleic acids
US4751211 *Aug 7, 1986Jun 14, 1988Aluminum Company Of AmericaComposite adsorbent for removing acids from organophosphate functional fluids
US4767670 *Jan 21, 1987Aug 30, 1988E. I. Du Pont De Nemours And CompanyChromatographic supports for separation of oligonucleotides
US4804561 *Oct 9, 1987Feb 14, 1989Chisso CorporationProcess for producing ferromagnetic metal fine particles
US4824712 *Oct 8, 1985Apr 25, 1989Ppg Industries, Inc.Treatment of glass to reduce venting during thermal treatment and a glass article made thereby
US4910148 *Dec 10, 1987Mar 20, 1990Dynal A. S.Magnetic separation of magnetized particles from biological fluids
US5039559 *Nov 22, 1989Aug 13, 1991Sang Jean VMethod of making magnetically attractable particles
US5055194 *Jul 28, 1989Oct 8, 1991University Of PennsylvaniaSupport for high performance liquid chromatography in a magnetically stabilized fluidized bed
US5057426 *Nov 23, 1987Oct 15, 1991Diagen Institut Fur Molekular-Biologische, Diagnostik GmbhMethod for separating long-chain nucleic acids
US5075430 *Dec 18, 1990Dec 24, 1991Bio-Rad Laboratories, Inc.Process for the purification of DNA on diatomaceous earth
US5076950 *Jun 5, 1990Dec 31, 1991Syntex (U.S.A.) Inc.Magnetic composition for particle separation
US5155018 *Jul 10, 1991Oct 13, 1992Hahnemann UniversityProcess and kit for isolating and purifying RNA from biological sources
US5206568 *May 10, 1991Apr 27, 1993Beckman Instruments, Inc.Coordinated control of stepper motors
US5210015 *Aug 6, 1990May 11, 1993Hoffman-La Roche Inc.Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
US5217804 *Nov 6, 1990Jun 8, 1993Eastman Kodak CompanyMagnetic particles
US5234809 *Jul 1, 1991Aug 10, 1993Akzo N.V.Process for isolating nucleic acid
US5236623 *Dec 12, 1990Aug 17, 1993Rhone-Poulenc ChimieProcess for the production of a silica colloid
US5279936 *Dec 22, 1989Jan 18, 1994Syntex (U.S.A.) Inc.Method of separation employing magnetic particles and second medium
US5312485 *Apr 15, 1993May 17, 1994J. M. Huber CorporationPrecipitated encapsulated paper pigments and methods
US5316699 *Mar 28, 1990May 31, 1994The United States Of America As Repesented By The Secretary Of CommerceProcess for the controlled preparation of a composite of ultrafine magnetic particles homogeneously dispersed in a dielectric matrix
US5340393 *Oct 12, 1993Aug 23, 1994E. I. Du Pont De Nemours And CompanyProcess for preparing silica coated inorganic particles
US5346994 *Jan 28, 1992Sep 13, 1994Piotr ChomczynskiShelf-stable product and process for isolating RNA, DNA and proteins
US5352645 *Nov 19, 1992Oct 4, 1994E. I. Du Pont De Nemours And CompanySilica microspheres, method of improving attrition resistance and use
US5368933 *Apr 19, 1993Nov 29, 1994Toda Kogyo Corp.Superparamagnetic fine particles of iron oxide and magnetic recording media containing said particles
US5389482 *Apr 23, 1992Feb 14, 1995Toda Kogyo Corp.Magnetic particle powder included in magnetic toners for magnetic image character recognition
US5395498 *Nov 2, 1992Mar 7, 1995Gombinsky; MosheMethod for separating biological macromolecules and means therfor
US5438127 *Sep 27, 1993Aug 1, 1995Becton Dickinson And CompanyDNA purification by solid phase extraction using a PCl3 modified glass fiber membrane
US5443791 *Aug 7, 1992Aug 22, 1995Perkin Elmer - Applied Biosystems DivisionAutomated molecular biology laboratory
US5458813 *Jul 21, 1993Oct 17, 1995Enichem S.P.A.Method for preparing boron-containing porous gels
US5470660 *Oct 5, 1993Nov 28, 1995Toda Kogyo CorporationIron oxide particles and process for producing the same
US5487972 *Jan 5, 1993Jan 30, 1996Hoffmann-La Roche Inc.Nucleic acid detection by the 5'-3'exonuclease activity of polymerases acting on adjacently hybridized oligonucleotides
US5503816 *Sep 27, 1993Apr 2, 1996Becton Dickinson And CompanySilicate compounds for DNA purification
US5512332 *Apr 22, 1994Apr 30, 1996Immunivest CorporationProcess of making resuspendable coated magnetic particles
US5512405 *Jun 7, 1995Apr 30, 1996Toda Kogyo CorporationIron oxide particles and process for producing the same
US5520899 *Apr 20, 1995May 28, 1996Becton, Dickinson And CompanyProcess for preparing boron, aluminum, and phosphorus silicates for purification of DNA
US5578238 *Apr 13, 1994Nov 26, 1996Lord CorporationMagnetorheological materials utilizing surface-modified particles
US5582988 *Sep 15, 1994Dec 10, 1996Johnson & Johnson Clinical Diagnostics, Inc.Methods for capture and selective release of nucleic acids using weakly basic polymer and amplification of same
US5597531 *Aug 22, 1989Jan 28, 1997Immunivest CorporationResuspendable coated magnetic particles and stable magnetic particle suspensions
US5599627 *Oct 4, 1994Feb 4, 1997Toda Kogyo CorporationMagnetic particles comprising magnetite core and process for producing the same
US5610274 *Sep 28, 1992Mar 11, 1997Cpg, Inc.Production and use of magnetic porous inorganic materials
US5648170 *Apr 20, 1994Jul 15, 1997Toda Kogyo CorporationCoated granular magnetite particles and process for producing the same
US5658548 *Jun 7, 1995Aug 19, 1997Promega CorporationNucleic acid purification on silica gel and glass mixtures
US5660984 *Dec 9, 1994Aug 26, 1997Davis; Thomas E.DNA isolating apparatus comprising a non-porous DNA binding, anion exchange resin and methods of use thereof
US5662824 *Nov 29, 1994Sep 2, 1997Alfa Biotech SpaMagnetically attractable particles and method
US5665554 *Jun 9, 1995Sep 9, 1997Amersham International PlcMagnetic bead precipitation method
US5681946 *Apr 3, 1996Oct 28, 1997Amersham International PlcPrecipitating polymers
US5683875 *May 4, 1995Nov 4, 1997Hewlett-Packard CompanyMethod for detecting a target nucleic acid analyte in a sample
US5693502 *Jun 7, 1995Dec 2, 1997Nexstar Pharmaceuticals, Inc.Nucleic acid ligand inhibitors to DNA polymerases
US5693785 *Nov 17, 1994Dec 2, 1997Becton, Dickinson And CompanyPurification of DNA on Hydroxylated Silicas
US5698271 *Jun 7, 1995Dec 16, 1997Immunivest CorporationMethods for the manufacture of magnetically responsive particles
US5705137 *May 29, 1996Jan 6, 1998Degussa AktiengesellschaftPrecipitated silicas, a process for their preparation and their use in vulcanizable rubber mixtures
US5723591 *Nov 15, 1995Mar 3, 1998Perkin-Elmer CorporationSelf-quenching fluorescence probe
US5734020 *Jun 2, 1995Mar 31, 1998Cpg, Inc.Production and use of magnetic porous inorganic materials
US5747663 *Feb 3, 1995May 5, 1998Qiagen GmbhProcess for the depletion or removal of endotoxins
US5763173 *Jun 7, 1995Jun 9, 1998Nexstar Pharmaceuticals, Inc.Nucleic acid ligand inhibitors to DNA polymerases
US5783686 *Sep 15, 1995Jul 21, 1998Beckman Instruments, Inc.Method for purifying nucleic acids from heterogenous mixtures
US5804375 *Apr 25, 1995Sep 8, 1998Roche Molecular Systems, Inc.Reaction mixtures for detection of target nucleic acids
US5898071 *Jan 2, 1998Apr 27, 1999Whitehead Institute For Biomedical ResearchDNA purification and isolation using magnetic particles
US5904848 *Feb 21, 1996May 18, 1999Cpg, Inc.Controlled pore glass-synthetic resin membrane
US5925573 *Mar 20, 1996Jul 20, 1999Bio MerieuxMethod and device for the determination of an analyte using super paramagnetic reactive particles
US5928958 *Jul 27, 1995Jul 27, 1999Pilgrimm; HerbertSuperparamagnetic particles, process for their manufacture and usage
US5945525 *Jul 8, 1996Aug 31, 1999Toyo Boseki Kabushiki KaishaMethod for isolating nucleic acids using silica-coated magnetic particles
US5972721 *Mar 14, 1997Oct 26, 1999The United States Of America As Represented By The Secretary Of The Air ForceImmunomagnetic assay system for clinical diagnosis and other purposes
US5990301 *Feb 3, 1995Nov 23, 1999Qiagen GmbhProcess for the separation and purification of nucleic acids from biological sources
US5990479 *Nov 25, 1997Nov 23, 1999Regents Of The University Of CaliforniaOrgano Luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes
US6027945 *Jan 21, 1997Feb 22, 2000Promega CorporationMethods of isolating biological target materials using silica magnetic particles
US6136083 *May 10, 1996Oct 24, 2000Institut Fuer Neue Materialien Gemeinnuetzige GmbhCoated inorganic pigments, process for their production and their use
US6255477 *Jun 6, 1996Jul 3, 2001Roche Diagnostics GmbhParticles having a magnetic core and outer glass layer for separating biological material
US6274386 *Jun 6, 1997Aug 14, 2001Roche Diagnostics GmbhReagent preparation containing magnetic particles in tablet form
US6296937 *Jan 8, 2001Oct 2, 2001W. R. Grace & Co.-Conn.Silica adsorbent on magnetic substrate
US6368800 *Dec 13, 1999Apr 9, 2002Promega CorporationKits for isolating biological target materials using silica magnetic particles
US6545143 *Nov 23, 1999Apr 8, 2003Roche Diagnostics, GmbhMagnetic particles for purifying nucleic acids
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US7249521Aug 24, 2005Jul 31, 2007Peter FrascaMethod for determining the presence of a magnetically susceptible or magnetic material in a sample
US8865000May 27, 2011Oct 21, 2014Basf SeUtilization of the naturally occurring magnetic constituents of ores
US20040126902 *Jun 27, 2003Jul 1, 2004Toyo Boseki Kabushiki KaishaMagnetic carrier for biological substance, production method thereof and isolation method of biological substance using same
US20090258368 *Apr 14, 2009Oct 15, 2009Noonan John MParamagnetic nucleation nanoparticles
US20100041034 *Apr 14, 2009Feb 18, 2010Murante Richard SMethod for manipulating samples with magnetic nucleation nanoparticles
US20100062421 *Apr 25, 2008Mar 11, 2010Wensheng XiaCompositions, methods, and devices for isolating biological materials
US20120258553 *Dec 16, 2010Oct 11, 2012Koninklijke Philips Electronics N.V.Analyte measurement apparatus and method
EP1642648A1Aug 12, 2005Apr 5, 2006Boehringer Mannheim GmbhApparatus and method for regulating the temperature of a liquid
WO2009129236A2 *Apr 14, 2009Oct 22, 2009Integrated Nano-Technologies LlcMethod for manipulating samples with magnetic nucleation nanoparticles
WO2009129236A3 *Apr 14, 2009Apr 22, 2010Integrated Nano-Technologies LlcMethod for manipulating samples with magnetic nucleation nanoparticles
WO2009154852A2 *Apr 14, 2009Dec 23, 2009Integrated Nano-Technologies LlcParamagnetic nucleation nanoparticles
WO2009154852A3 *Apr 14, 2009Mar 4, 2010Integrated Nano-Technologies LlcParamagnetic nucleation nanoparticles
WO2013185078A1 *Jun 7, 2013Dec 12, 2013Somalogic, Inc.Aptamer-based multiplexed assays
Classifications
U.S. Classification435/6.12, 501/12, 436/526, 435/6.1
International ClassificationC23C30/00, C12Q1/68, C12M1/34, C23C26/00, C12N15/10, B01J19/00, C03B8/02, C01B33/18, H01F1/06, C03B19/10, C03C14/00, B03C1/01, H01F1/44, H01F1/00
Cooperative ClassificationH01F1/0054, C03B19/1065, H01F1/44, C03C1/006, C23C30/00, C12Q1/6816, C03C3/091, C12Q2561/101, C03C12/00, B82Y25/00, C03C2214/08, C23C26/00, B03C1/01, C03C3/093, C12Q1/6848, C12Q1/6806, C03C14/004, C12N15/1013, H01F1/0063, C03C2214/32
European ClassificationB82Y25/00, C12Q1/68D2A, C12Q1/68A4, C23C26/00, C23C30/00, H01F1/00E10M, H01F1/44, C12N15/10A2D, B03C1/01
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Jan 2, 2003ASAssignment
Owner name: ROCHE DIAGNOSTICS GMBH, GERMANY
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Effective date: 20021112