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WO1999063109A1 - Procede permettant de determiner des antigenes - Google Patents

Procede permettant de determiner des antigenes Download PDF

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Publication number
WO1999063109A1
WO1999063109A1 PCT/AT1999/000139 AT9900139W WO9963109A1 WO 1999063109 A1 WO1999063109 A1 WO 1999063109A1 AT 9900139 W AT9900139 W AT 9900139W WO 9963109 A1 WO9963109 A1 WO 9963109A1
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WIPO (PCT)
Prior art keywords
nucleic acid
standard
antigen
ligand
determined
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PCT/AT1999/000139
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German (de)
English (en)
Inventor
Klaus Zimmermann
Peter Turecek
Hans-Peter Schwarz
Jürgen SIEKMANN
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Baxter Aktiengesellschaft
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Publication date
Application filed by Baxter Aktiengesellschaft filed Critical Baxter Aktiengesellschaft
Priority to EP99923299A priority Critical patent/EP1082464A1/fr
Priority to AU40226/99A priority patent/AU746812B2/en
Priority to JP2000552301A priority patent/JP2002517206A/ja
Publication of WO1999063109A1 publication Critical patent/WO1999063109A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification

Definitions

  • the invention relates to a method for determining antigens in a sample by amplifying a nucleic acid which is bound to the antigen to be determined.
  • antigens to be detected have to be determined precisely in low concentration ranges or amounts in order to obtain important information, for example on clinical pictures, biological contaminations, physiological values of selected proteins, etc.
  • Antigens are usually determined by antibody tests, for example by means of ELISA.
  • the antigen to be determined is detected by binding to an antibody specific for the antigen.
  • the level of bound antibody / antigen is then usually determined by a proven detection method, such as Radioactivity, fluorescence, staining reaction, etc., preferably using secondary antibodies.
  • a pathogen is the antigen responsible for scrapie.
  • Scrapie was first discovered in sheep about 250 years ago.
  • diseases related to scrapie have also been described in other animals and also in humans as transmissible spongiform encephalopathy (TSE, transmissible spongiform encephalopathies).
  • TSE transmissible spongiform encephalopathy
  • Prions are believed to be the cause of these diseases.
  • Prion diseases cannot be diagnosed satisfactorily with conventional antigen / antibody tests.
  • a prion protein is either not infectious, one speaks then of a PrP c , or else infectious, then one speaks of a PrP Sc .
  • the non-infectious prion protein PrP c is an ubiquitous cellular protein which, due to a change in conformation, becomes the infectious prion protein PrP Sc .
  • a proteinase K digest is usually carried out with a subsequent western blot, the infectious prion protein PrP Sc proving to be partially resistant to the proteinase K digest.
  • Korth et al. (Nature 390 (1998), 74-77) describe a process for the production of antibodies which specifically recognize either the “normal”, non-infectious prion protein or which specifically recognize the infectious prion protein.
  • the prions mostly cause progressive diseases of the central nervous system (spongiform encephalopathies), for example scrapie in sheep, Kuru, RSE in cattle as well as Creutzfeld-Jakob disease (CJD), Gerstmann-St Hurssler syndrome (GSS) and fatal familial insomnia (FFI ).
  • the prions are characterized by special physicochemical and biological properties against viruses and bacteria: high resistance to inactivation by formaldehyde, nucleases, heat, UV and ionizing rays, and are not recognizable as virions or cells in the electron microscope. They are insensitive to interferons, show no interference with viruses and, moreover, no nucleic acid has been detected to date.
  • nvCJD neuropathological features
  • prion tests such as Western blot using specific antibodies, have insufficient sensitivity to be able to determine prion protein effectively.
  • WO 97 37 227 describes an immunohistochemical test for the determination of prions in tissues. Using this method, the modified prion protein (PrP Sc ) can be determined in histological sections from infected tissue parts. However, this method is not very reliable and is particularly unsuitable for the examination of blood and blood products.
  • PCR polymerase chain reaction
  • connection molecule with bispecific binding affinity for DNA and antibodies, eg a chimeric molecule made of streptavidin for biotinylated DNA and protein A for immunoglobulin G, is used to bind a specific DNA molecule (marker) unspecifically to an antigen antibody -Binding complex, so that an antigen-antibody-linker-DNA complex is formed.
  • the bound DNA marker can now be primed in a PCR be amplified. The detection of the PCR product indicates the presence of the antigens.
  • the labeled DNA-antibody complexes are assembled in situ in the reaction mixture, which can lead to different stoichiometry in the composition and binding of the DNA. Additional steps for adding biotinylated reagents and binding proteins as well as numerous washing steps are required to remove excess reagents. The many process steps make this process complicated and time-consuming in order to be able to use it in routine analysis. Furthermore, this method is not suitable for complex mixtures of different antigens.
  • Hendrickson et al. (Nucl. Acids Res. 23 (3) (1995), 522-529) therefore describe an improved immunoassay for the simultaneous determination of different analytes.
  • the antibodies and the DNA in this case a single-stranded oligonucleotide, were activated separately and coupled directly to one another in a spontaneous reaction.
  • Hendrickson et al. describes the simultaneous detection of three different analytes by means of immuno-PCR.
  • the DNA-labeled antibodies that are bound to the corresponding antigen are amplified and determined by means of PCR.
  • the object of the present invention is therefore to provide a reliable and sensitive method for determining antigens, with which above all false-negative results can be excluded from the outset.
  • the object of the invention is achieved by a method for determining antigens by amplifying a nucleic acid (target nucleic acid) which is bound to a ligand specific for the antigens, which is characterized in that the determination is carried out in the presence of at least one internal standard .
  • antigen determinations can be monitored and controlled in an elegant manner by means of immune amplification reactions. Above all, false negative results can be reliably excluded by the method according to the invention. Furthermore, by using internal standards, it is also possible to obtain a quantitative reference value for the antigen determination in the sample, especially if more than one standard is used. In routine operation, however, it will generally not be necessary to use more than one standard, especially if the use of a standard is only intended to rule out false-negative results.
  • a nucleic acid bound to a ligand is preferably used as the standard (standard nucleic acid).
  • Standard nucleic acids which differ from the nucleic acid with which the (target) antigen is determined (target nucleic acid), at least in one detectable feature, are particularly preferably used.
  • both the standard nucleic acid and the target nucleic acid are then amplified and the amplified nucleic acids are examined.
  • examples of (gene) amplification methods are reverse transcriptase PCR (RT- PCR), the ligase chain reaction (LCR) and the nucleic acid sequence based amplification (NASBA).
  • PCR polymerase chain reaction
  • RT- PCR reverse transcriptase PCR
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence based amplification
  • the "Taqman assay” combines the polymerase chain reaction with hybridization of the target sequence with a labeled sample (Holland et al., Proc. Natl. Acad. Sei., 88 (1991), 7276- 7280).
  • the standard nucleic acid can preferably have a length of at least 70 nucleic acids. A length of> 100 to several hundred nucleic acids is preferably selected.
  • the target nucleic acid is bound to the ligand, specific to the antigen to be determined.
  • Antibodies which are specific for the antigen to be determined are preferably used as ligands.
  • affinity ligands can also be used, e.g. Peptides that bind specifically to the antigen.
  • the standard nucleic acid usually differs from the target nucleic acid in at least one detectable feature, but is preferably amplified using the same means as primers. Standard nucleic acids that have a different size than the nucleic acid to be determined or a unique restriction interface have proven to be preferred, since the result can be evaluated by simple gel electrophoresis.
  • Preferred standards differ from the nucleic acid to be determined in 1% to 20% of their length or by at least 3, at most 50 nucleotides, a standard nucleic acid which is longer than the target nucleic acid as "plus” (“+”) Standard is referred to, and a standard nucleic acid that is smaller than the target nucleic acid is referred to as the "minus" ("-”) standard.
  • the exact sequence of the standard nucleic acid should of course be known.
  • Both the nucleic acid for the internal standard and the nucleic acid which is bound to the ligand, specifically for the antigen to be determined, can be used either as double-stranded or as single-stranded nucleic acid.
  • Single-stranded DNA with a terminal amino group is preferably selected for chemical crosslinking with the specific ligand.
  • the internal standard can also be provided as a combination of the standard nucleic acid with a ligand, specific for a standard antigen.
  • the internal standard can now either be bound directly to the specific ligand or else by means of a connecting molecule (linker) which has a DNA binding site on one side, for example streptavidin, and on the other side a specific ligand which is active against an antigen (Standard antigen), the connection between the specific ligand and the DNA is made.
  • the DNA is bound directly to the ligand by chemical binding, for example by means of affinity binding or covalent binding.
  • Peptides or monospecific antibodies can be used as ligands for the internal standard and / or for the antigen to be determined.
  • the ligands that are bound to the standard nucleic acid can also be specifically directed against the antigen to be determined in order to act competitively with the target ligands specifically for the antigen to be determined.
  • Polyspecific antibodies that recognize the standard antigen can also be used.
  • Ligands which are specific for the antigen to be determined are preferably antibodies which are suitable for the determination of modified proteins and prion proteins.
  • ligands can also be selected for other purposes specifically for the antigens to be determined, e.g. from the group of viral, bacterial or human proteins. Specific antibodies for a prion protein are preferably used in the determination of prions.
  • the primers used in the amplification can preferably contain markers which further increase the detection limit for determining the amplified nucleic acid, for example fluorescent or radioactive groups or chemical groups which can be detected using affine proteins and downstream detection reactions (for example biotin-avidin, DIG Labeling etc.), with primers with fluorescent groups being particularly preferred.
  • markers which further increase the detection limit for determining the amplified nucleic acid for example fluorescent or radioactive groups or chemical groups which can be detected using affine proteins and downstream detection reactions (for example biotin-avidin, DIG Labeling etc.), with primers with fluorescent groups being particularly preferred.
  • the detection of the amplified nucleic acids can be carried out in a wide variety of ways, but usually a step is to be provided in which the amplified standard nucleic acid is separated from the amplified target nucleic acid and the separated nucleic acids are determined separately.
  • This separation step preferably consists of a gel electrophoresis or a chromatographic process.
  • Antigens that can be determined by the method according to the invention include antigens from biological samples from mammals, such as body fluids, e.g. Plasma, blood, blood cells and serum, tissue, but also antigens from eukaryotic cell cultures.
  • antigens of pathogens or marker antigens for pathogens are preferably determined. These antigens include viral antigens, bacterial antigens and, in particular, antigens for which no routine detection method has hitherto been available, such as modified ones
  • modified proteins such as prions, which are associated with infectious diseases of the central nervous system, such as CJD and BSE, or other pathogens of TSE.
  • modified proteins can be obtained by induced conformational changes.
  • This conformational change on proteins, such as possibly recombinant PrPc can be brought about by previous incubation with infectious molecules (such as PrPSc), which then e.g. can be detected by radioactive markers.
  • infectious molecules such as PrPSc
  • one or more of the antigens of human pathogens are determined.
  • the antigens to be determined can be in a purified protein-containing preparation, for example a plasma fraction, a preparation of a purified nigen plasma protein, or a biological drug. However, they can also be reliably determined in plasma pools or other complex mixtures or in the biological samples mentioned. Quality assurance as described in W096 / 35437 can also be carried out appropriately adapted with the method according to the invention.
  • the corresponding internal standard can be added before sample preparation, during all intermediate stages or only immediately before the amplification reaction.
  • the standard is added from the start as an unbound nucleic acid or as a bound nucleic acid after the immune complex formation.
  • two (or more) different nucleic acids can be added as an internal standard.
  • the internal standard is preferably used in a concentration just above the detection limit.
  • the antigen that is bound (recognized) by the internal standard can be added to the sample to be determined (e.g. the antigen determination approach), or it can already be present in the sample in a natural way (a priori).
  • the results obtained with the method according to the invention can usually be interpreted as follows: i) no detection of the internal standard (e.g. no visible band): the determination has e.g. does not work due to the amplification reaction (e.g. the PCR); this can rule out false negative results. ii) only the internal standard is detectable (e.g. only the standard band is visible): the determination including the amplification reaction (e.g. the PCR) worked, the sample is negative; iii) Standard and sample nucleic acids are detectable (e.g. both bands are visible): positive sample.
  • Suitable reaction vessels for immuno-PCR should preferably have a good protein binding capacity for immobilizing the Own proteins.
  • the tubes for the PCR must also be thermostable.
  • Polystyrene is the most suitable material in terms of protein binding, but no such PCR-compatible plates have been sold commercially until now. According to the invention, however, polycarbonate sheets can be used as replacements. It is also conceivable to use chemically modified materials for the covalent binding of proteins or materials already coated with antibodies.
  • the plates are preferably coated overnight at 4 ° C. Higher temperatures of up to 37 ° C. with a shorter incubation time are also possible.
  • PBS saline solution buffered in phosphate
  • any other common buffer e.g. Polycarbonate buffer.
  • the blocking can be carried out, for example, with PBS / 1% BSA (bovine serum albumin). Any other suitable reagent, e.g. Milk powder or pre-made blocking reagents can be used.
  • BSA bovine serum albumin
  • Incubation with the ligand / DNA complex preferably takes place for half an hour at room temperature, but other time periods (preferably 5 minutes to 2 hours) and other temperatures (preferably 4 ° to 37 ° C.) are also possible.
  • Example 1 The optimal reaction conditions for the PCR are described in Example 1. A large number of changes are of course possible (in particular changes in the number of cycles, incubation times, temperature profile, use of different polymerases, amplification systems, primer strategies, etc.).
  • the optimal conditions can, e.g. the protocol used in the examples is not optimal, for the individual antigens to be determined are readily determined by the person skilled in the art in the present field.
  • Another aspect of the present invention relates to the use of the method according to the invention for checking and Quality assurance of biological preparations.
  • the safety, in particular the virus safety, of stable blood products depends in principle on the extent of the contamination of pathogens, in particular on the virus contamination of the starting material, on the depletion capacity of the process and on the specific virus inactivation stages in the course of the manufacturing process.
  • An important criterion of quality assurance is therefore to exclude positive starting material from processing into pharmaceutical drugs.
  • new quality criteria can be determined for biological products which are defined by an extremely low defined or missing content of contaminating antigens.
  • Another aspect of the invention is a reagent for use in the method of the invention, which a) a reactant bound to a ligand that is specific for the antigen to be determined, and b) a reactant as an internal standard that is different from the reactant differs from a) in the reaction, contains.
  • nucleic acids or enzymes can be used as reactants in order, for example, to obtain a controlled enzyme reaction.
  • nucleic acids are used as reactants, which have a detectable feature, e.g. differ in length.
  • the target nucleic acid is preferably bound to an antibody which is specific for the modified protein.
  • the standard nucleic acid is bound to a ligand that, especially on a ligand that is already bound to a standard antigen.
  • the standard antigen can be the same as the antigen to be determined (competitive immune reaction).
  • the standard nucleic acid in the reagent according to the invention is preferably bound to a ligand which is specific for an antigen different from the antigen to be determined (target antigen).
  • the ligand / DNA complexes can be stored at -20 ° C. or 4 ° C. in a suitable buffer, preferably PBS. They are then stable for several months.
  • This preparation corresponds to a commercial form.
  • the optimal concentration at which the complexes are used is approximately 10,000 to approximately 1,000,000 copies per well of a microtiter plate. Preferably, fewer than 1 million copies are used to avoid non-specific binding in the wells.
  • Conventional formulation reagents such as albumin as carrier protein, surfactants, etc. can be used to prepare the reagent according to the invention.
  • the present invention also relates to the use of the method according to the invention for the quantification of antigens, in particular for the quantification of pathogens in mammalian samples or of human pathogens in biological medicinal products.
  • Fig. 1 shows the schematic representation of the IMMUNO-PCR with internal standard.
  • a microtiter plate is coated with an antibody specific for a standard antigen and an antibody specific for the antigen to be determined. From these The standard antigen and the antigen to be determined are bound to the body. The ligands are then bound to the antigens.
  • Example 1 Add a DNA standard to the PCR
  • the PCR part of the immuno-PCR was checked according to the invention for false negative reactions by adding a standard nucleic acid which differs in length from the nucleic acid of the antibody / nucleic acid conjugate specific to an antigen.
  • a cellular (non-infectious) prion protein (PrP c ) is used as the antigen.
  • This prion protein is not in a purified form, but is in a homogenate which is obtained from a mouse brain.
  • the brain was homogenized in a buffer (0.1 g / ml) consisting of a 0.32 M cane sugar solution, 0.5% deoxycholate and 0.5% NP-40. After a centrifugation step (15 min at 4500 rpm) the pellet was discarded and then the amount of protein in the supernatant was determined.
  • the PCR solution (50 ⁇ l) contained 1 U of the polymerase AmpliTaq Gold TM (Perkin Elmer, Norwalk, CT, USA), 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl 2 , 0.001% gelatin, 200 ⁇ M of each dNTP and 500 ng each of the corresponding primers IP1 / IP2 (see Table 1).
  • the samples were overlaid with 50 ⁇ l of mineral oil and initially incubated for 14 min at 94 ° C. to activate the polymerase.
  • samples 1-6 1000 copies of the standard nucleic acid IPOS1 were added, in samples 7-12, 100 copies of IPOS1 were added. Coating was carried out with either 8 or 0.8 ⁇ g homogenate.
  • a standard nucleic acid is not added to the PCR, but a marker antigen is added at the beginning of the experiment as an internal standard in an amount that can still be determined at the same time put the specific antigen on the appropriate wells of a microtiter plate and incubate overnight at 4 ° C. This ensures that all disruptive influences that could lead to a false negative reaction (this also includes, for example, the inadvertent failure to coat a recess) are also checked.
  • incubation is then carried out simultaneously with a corresponding standard antibody / DNA complex and subsequently amplified with the same pair of primers.
  • the wells of the microtiter plate must be coated accordingly with an antibody for the antigen to be detected and an antibody for the standard antigen.
  • the standard antigen may also occur naturally, such as certain proteins (e.g. Protein C) when using biological fluids such as e.g. Plasma.
  • An antibody / DNA conjugate was produced by covalently binding an oligonucleotide to a monoclonal anti-prion antibody in accordance with Hendricksson et al.
  • An amino-modified reporter oligonucleotide IPl, manufactured by Metabion, Martinsried, Germany
  • the monoclonal anti-prion antibody 6H4 (Prionix, Basel, Switzerland) were used as the starting material for producing the conjugate.
  • Step 1 preparation of the acetylthioacetyl-derivatized DNA
  • the acetylthioacetyl-derivatized DNA is prepared by reaction with succinimidyl-S-acetylthioacetate (SATA reagent / Pierce, Rockford, IL, USA).
  • each 20 ⁇ l of a solution of the IPOl oligonucleotide in H 2 0 was 2.3 ⁇ l of a sodium carbonate buffer (300 mM, pH 9.0) and a solution of 115 mM SATA placed in DMF and incubated for 30 minutes at room temperature with gentle shaking.
  • the mixture was then buffered over a HiTrap TM column (1.6 ⁇ 2.5 cm, Sephadex G-25 superfine / Pharmacia, Uppsala, Sweden) using an FPLC system (from Pharmacia).
  • Step 3 preparation and purification of the DNA-antibody conjugate
  • the derivatives prepared in step 1 and step 2 were combined (in each case approximately 1.5 ml of solution of the acetylthioacetyl-derivatized DNA or of the maleimide-modified antibody).
  • the coupling reaction had started by adding 2 ⁇ l of a 1 M aqueous hydroxylamine hydrochloride solution, pH 7.0, containing 50 mM EDTA, the mixture was incubated for 2 hours at room temperature with the exclusion of light and gentle shaking. The reaction was then stopped by adding 2 .mu.l of a 10 mM solution of N-ethylmaleimide (from Pierce) in DMF.
  • the reaction mixture was concentrated using a Centricon 3 concentrator (Amicon, Beverly, MA, USA) by centrifugation at 7000 xg.
  • the concentrated reaction mixture (approx. 500 ⁇ l) was then purified by gel filtration using the Pharmacia FPLC system (Pharmacia, Uppsala, Sweden).
  • fractions at the elution maximum were collected and used for immuno-PCR in various dilutions.

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Abstract

L'invention concerne un procédé permettant de déterminer des antigènes dans un échantillon par l'amplification d'un acide nucléique qui est lié à un ligand spécifique aux antigènes. Cette détermination est effectuée en présence d'au moins un standard interne.
PCT/AT1999/000139 1998-06-04 1999-06-02 Procede permettant de determiner des antigenes WO1999063109A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP99923299A EP1082464A1 (fr) 1998-06-04 1999-06-02 Procede permettant de determiner des antigenes
AU40226/99A AU746812B2 (en) 1998-06-04 1999-06-02 Method for determining antigens
JP2000552301A JP2002517206A (ja) 1998-06-04 1999-06-02 抗原を決定するための方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AT0096398A AT407160B (de) 1998-06-04 1998-06-04 Verfahren zur bestimmung von antigenen
ATA963/98 1998-06-04

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WO1999063109A1 true WO1999063109A1 (fr) 1999-12-09

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EP (1) EP1082464A1 (fr)
JP (1) JP2002517206A (fr)
AT (1) AT407160B (fr)
AU (1) AU746812B2 (fr)
WO (1) WO1999063109A1 (fr)

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WO2001031056A3 (fr) * 1999-10-27 2002-06-27 Univ Liege Methode de detection par pcr
GB2375822A (en) * 2000-12-05 2002-11-27 Norchip As Immunoassay using nucleic acid amplification and real-time measurement
EP1270738A1 (fr) * 2001-06-18 2003-01-02 chimera biotec GmbH Méthode pour détecter des substances dans des liquides
WO2002016635A3 (fr) * 2000-08-25 2003-06-05 Friz Biochem Gmbh Procede pour le marquage de substances chimiques
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Cited By (16)

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WO2001031056A3 (fr) * 1999-10-27 2002-06-27 Univ Liege Methode de detection par pcr
WO2002016635A3 (fr) * 2000-08-25 2003-06-05 Friz Biochem Gmbh Procede pour le marquage de substances chimiques
US7910294B2 (en) 2000-12-05 2011-03-22 Norchip A/S Ligand detection method
GB2375822A (en) * 2000-12-05 2002-11-27 Norchip As Immunoassay using nucleic acid amplification and real-time measurement
WO2002046464A3 (fr) * 2000-12-05 2003-09-12 Norchip As Procede de detection de ligands
GB2375822B (en) * 2000-12-05 2005-04-06 Norchip As Real time ligand detection comprising isothermal amplification
EP1395805A2 (fr) * 2001-06-11 2004-03-10 Illumina, Inc. Techniques de detection multiplexees
EP1395805A4 (fr) * 2001-06-11 2005-03-09 Illumina Inc Techniques de detection multiplexees
EP1270738A1 (fr) * 2001-06-18 2003-01-02 chimera biotec GmbH Méthode pour détecter des substances dans des liquides
US7932060B2 (en) 2003-04-18 2011-04-26 Becton, Dickinson And Company Immuno-amplification
US8372605B2 (en) 2003-04-18 2013-02-12 Becton, Dickinson And Company Immuno-amplification
US9499858B2 (en) 2003-04-18 2016-11-22 Becton, Dickinson And Company Immuno-amplification
US8304192B2 (en) 2004-03-05 2012-11-06 University Of Toledo Methods and compositions for assessing nucleic acids and alleles
US7476502B2 (en) 2004-04-12 2009-01-13 Willey James C Methods and compositions for assaying analytes
EP1745157A4 (fr) * 2004-04-12 2008-06-11 Ohio Med College Methodes et compositions pour analyser des analytes
EP1745157A2 (fr) * 2004-04-12 2007-01-24 The Medical College Of Ohio Methodes et compositions pour analyser des analytes

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AU4022699A (en) 1999-12-20
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AT407160B (de) 2001-01-25
JP2002517206A (ja) 2002-06-18
ATA96398A (de) 2000-05-15

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