WO1987004794A1 - Agglutination de latex a l'aide d'un systeme d'avidine/biotine - Google Patents

Agglutination de latex a l'aide d'un systeme d'avidine/biotine Download PDF

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Publication number
WO1987004794A1
WO1987004794A1 PCT/US1987/000245 US8700245W WO8704794A1 WO 1987004794 A1 WO1987004794 A1 WO 1987004794A1 US 8700245 W US8700245 W US 8700245W WO 8704794 A1 WO8704794 A1 WO 8704794A1
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Prior art keywords
reagent
analyte
interest
antibody
particle
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PCT/US1987/000245
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English (en)
Inventor
David A. Fuccillo
Isabel C. Shekarchi
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Microbiological Associates, Inc.
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Application filed by Microbiological Associates, Inc. filed Critical Microbiological Associates, Inc.
Publication of WO1987004794A1 publication Critical patent/WO1987004794A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex

Definitions

  • the present invention relates to an agglutination test and reagent for detecting the presence of an analyte of interest, e.g., an antigen or antibody, within a sample specimen.
  • an analyte of interest e.g., an antigen or antibody
  • agglutination test which is currently in use, called a latex agglutination test, utilizes a suspension of plastic particles or beads carried in an aqueous vehicle. Describing a typical latex agglutination test as it would be performed for the detection of an antibody, a reagent is prepared for the latex suspension by coating the beads or particles in the suspension with a ligand, for example an antigen specific for the tested-for antibody. The reagent so prepared is then mixed, as on a slide, with a diluted or undiluted serum specimen.
  • a ligand for example an antigen specific for the tested-for antibody
  • Antibody present in the serum sample or specimen attaches to the antigen coated on the beads or particles, resulting in agglutination of the beads or particles which may be visually observed, indicating a positive test. In a negative test, agglutination does not occur and the mixture of specimen and reagent remains smooth and milky in appearance.
  • An agglutination test as just described may be quickly performed and is relatively simple to carry out, which has obvious advantages.
  • the maximum adsorption -- and therefore the maximum test sensitivity -- requires the use of ultrapure antibody, which is usually difficult to obtain. Additionally, impurities present in the antibody preparation may compete with the antibody for adsorption sites on the latex particle surface.
  • crosslinking or bridging with chemicals such as glutaraldehyde may be used. Chemicals such as these, however, may inactivate the ligand to the extent that it may no longer function to bind another ligand, e.g., an antigen or antibody. For these and other reasons, relatively few latex agglutination tests are available for viral antibody or antigen detection.
  • Agglutination tests are also known which rely upon cellular bodies, e.g., treated red blood cells, as the centers of cell aggregates formed in the agglutination.
  • An assay performed with treated blood cells involves problems such as maintaining a proper supply of the processed cells, shelf-lite problems, expense, etc.
  • U.S. Patent No. 4,434,150 (Azad et al.) discloses an immunological detecting reagent which comprises a water soluble polymer having an antibody or antigen attached via an avidin-biotin linkage.
  • the water soluble polymer also has a plurality of detectable markers attached to it.
  • U.S. Patent No. 4,228,237 discloses a method for determining the presence of a ligand in a liquid medium which utilizes a detectable enzyme complex to which is bound an antigen or antibody corresponding to the ligand. The binding is via the avidinbiotin system. The detectable enzyme complex is then contacted with an insoluble phase containing a specific binding substance for the ligand. The detectable enzyme complex binds with the specific binding substance and is then detected in the insoluble phase.
  • U.S. Patent No. 4,298,685 discloses a method for the quantitative determination of a biological substance in a test sample which comprises mixing a test sample containing an unknown amount of antigen with known amounts of biotin labelled antibody and antigen labelled with detectable enzyme.
  • the enzyme labelled antigen competes with the unknown amount of free antigen in the system for the biotin labelled antibody.
  • Two different antigen-antibody complexes result: one labelled with both enzyme and biotin and the second labelled only with biotin.
  • the antigen-antibody complexes are then contacted with an insoluble solid support coated with avidin.
  • the avidin binds with the biotin labelled complexes to form a precipitate comprising solid support-avidin-biotin-antibody-antigen. Enzyme activity is then measured in order to quantitate the biological substance.
  • U.S. Patent Nos. Re. 31,712 and 4,478,914 disclose methods for coating the surface of beads, particles, spheres etc. with biotin or caproylamidobiotin N-hydroxysuccinimide to form a multi-layered product having successive, repetitive layers of avidin-extender attached to the biotin.
  • U.S. Patent Nos. 4,141,965 and 4,210,622 (Soothill et al.) and U.S. Patent No. 4,331,649 (Chantler et al.) each disclose the estimation of an immune complex using a latex particle agglutination test.
  • the latex particles are coated with a constituent of the immune complex and the constituent is attached to the latex bead via a coupling agent such as dinitrophenol.
  • the coated latex beads are contacted with a sample containing an unknown amount of an antibody to the constituent. Immune complex is then measured by the inhibition of the agglutination reaction.
  • U.S. Patent No. 4,582,810 (Rosenstein) describes a diagnostic particle for use in an aqueous suspension in agglutination tests.
  • the particle comprises a carboxylated latex particle having avidin molecules linked thereto through covalent amide bonds. Biotin molecules are complexed with the avidin molecules and antibody molecules are linked through covalent amide bonds to a significant proportion of the biotin molecules.
  • the latex particle is coated with antibody and is useful for detecting an antigen specific for the antibody.
  • a disadvantage of such a diagnostic particle is that the formation of the covalent amide bonds between the latex particle and the avidin molecules requires additional steps which add to the time and expense of preparing such a particle and also introduces the possibility of deactivating antibody binding site(s).
  • a general object of the invention is to provide an improved agglutination test which obviates many of the problems and disadvantages associated with such tests as currently available.
  • a reagent for detecting an analyte of interest in a sample fluid comprises:
  • each such derivatized biotin molecule having the structure:
  • A is a ligand specific for the analyte of interest and -R- is a linker group which covalently joins A to biotin.
  • the present invention also provides a reagent for detecting an analyte of interest in a sample which comprises:
  • b a plurality of avidin molecules noncovalently attached to the surface of the molecule; and c. derivatized biotin hydrazide molecules complexed with at least a portion of the avidin molecules, each such derivatized biotin hydrazide molecule being covalently bound to a carbohydrate-containing ligand specific for the analyte of interest.
  • the present invention provides a reagent for detecting an analyte of interest in a sample, which comprises:
  • each such derivatized biotin molecule having the structure:
  • A is a ligand specific for the analyte of interest and -R- is a linker group which covalently joins A to biotin.
  • -R- may be represented by the structure:
  • n is an integer between 2 and about 14.
  • -R- may also be represented by the structure:
  • A comprises a carbohydrate and -R- is covalently linked to A by means of the double bond of -R-.
  • the derivatized biotin molecule has the following structure:
  • the particle may be polyacrylate, polystyrene or polycarbonate.
  • the particle may also be any other inert particle which is capable of being suspended in a liquid vehicle, e.g. water, such as an inorganic clay particle, e.g. bentonite, or a fixed or tanned red blood cell.
  • the avidin molecules may be passively adsorbed to the particle surface and at least 1 and up to 4 derivatized biotin molecules may be complexed with each avidin molecule.
  • the analyte of interest is an antibody or an antigen.
  • the antibody to be detected may be specific for a virus, e.g. herpes simplex virus or human immunodeficiency virus.
  • the analyte of interest is a hormone.
  • the ligand comprises an antibody, an antigen, or a hapten.
  • the antibody may be a monoclonal antibody.
  • the antigen may be a polysaccharide or a glycoprotein.
  • Another reagent for detecting an analyte of interest in a sample fluid comprises:
  • each such derivatized biotin hydrazide molecule being covalently bound to a carbohydrate-containing ligand specific for the analyte of interest.
  • the ligand may comprise a polysaccharide or a glycoprotein.
  • the carbohydrate groups of the ligand must be pretreated so as to form aldehyde groups which react with the biotin hydrazide.
  • the particle may be polyacrylate, polystyrene or polycarbonate.
  • the avidin molecules are passively adsorbed to the particle surface.
  • the analyte of interest may be specific for a virus, e.g. herpes simplex virus or human immunodeficiency virus.
  • the analyte of interest may be a hormone.
  • liquid suspensions comprising the reagents of the present invention in a suitable liquid medium, e.g., water.
  • Also provided is a method for detecting the presence of an analyte of interest in a sample which comprises contacting the sample with a reagent of the present invention, wherein the reagent comprises a ligand specific for the analyte of interest, so as to form a complex between the analyte of interest and the reagent, wherein the complex is in the form of a precipitate or agglutinate, and detecting the presence of the complex and thereby the presence of the analyte of interest.
  • the contacting may be effected on the surface of a glass slide and may be done manually or mechanically.
  • the precipitate or agglutinate may be detected visually, microscopically or photometrically.
  • the present invention provides an agglutination test which utilizes a suspension of solid particles as the core material in the aggregates that form during the agglutination procedure, and an avidin-biotin linking system employed for coating such particles with a ligand. Such particles react and combine with the analyte of interest, which results in an agglutination indicative of a positive test.
  • solid inert particles as exemplified by the plastic beads or particles that typify a latex suspension, are coated with a ligand through an avidin to biotin bond, wherein the avidin is attached to the particle surface.
  • the particles and the antigen may be subjected to a relatively gentle nondestructive pretreatment, after which their attachment is readily accomplished.
  • the attachment through the avidin-biotin bond is simple and specific. Since no harsh chemicals are used in the adsorption procedure, it is contemplated that almost any ligand may be bound to the surfaces of the particles in the suspension. Because of the strength and specificity of the bond, relatively low concentrations of the ligand are required for adsorption on the particles of the suspension.
  • the present invention provides that the particles of the suspension are coated with avidin, which is readily adsorbed onto the particle surfaces.
  • Avidin as is known in the art, has four binding sites for biotin.
  • avidin-coated particles have potential binding sites for a biotinylated antigen which is four times the number of avidin proteins attached to the particle surfaces.
  • the ligand employed in agglutination tests may be easily and nondestructively conjugated with biotin. Mixture and interaction of a biotinylated ligand and suspension of avidin-coated particles readily produces a reagent suspension for the determination, in an agglutination test, of the presence or absence of an analyte of interest specific for the ligand.
  • Avidin is a glycoprotein of approximately 68,000 molecular weight present in egg white, which has been found to have an extraordinary high affinity for the small molecular weight biotin. Thus, biotin and avidin interact specifically and under mild conditions to form a strong, stable interlinked complex. Avidin has four binding sites, each of which may bind to a biotin mole cule and this binding persists when avidin attaches to a structure such as the surface of an inert particle or bead. Certain analogs or derivatives of avidin suggest their use as a substitute for avidin. A typical example of an avidin analog is streptavidin, which appears to be a 60,000 molecular weight protein and like avidin has four binding sites for biotin and a binding affinity similar to its analog.
  • avidin derivatives applicable to the invention are such materials as succinyl avidin, ferritin avidin, and crosslinked avidin.
  • the term "avidin component” refers to avidin as well as its recognized analogs and derivatives. Avidin is readily available in highly purified form and readily attaches to the surface of an inert particle or bead.
  • Biotin also known as Vitamin H, has a molecular weight of about 274. Biotin easily conjugates with many proteinaceous materials without altering the biological activity of the protein. Thus, it is easily conjugated with an antigen.
  • Biotin derivatives and analogs are contemplated for use in the present invention.
  • examples of such materials are biocytin, biotin hydrazide, biotinyl-N-hydroxysucciniraide ester, and other long chain biotinyl molecules.
  • biotin component refers to biotin as well we its recognized analogs and derivatives.
  • Biotin hydrazide is a particularly useful biotin derivative contemplated for use in the present invention in that it may be conjugated to carbohydrate residues present in polysaccharides or glycoproteins. To achieve this conjugation the carbohydrate residues are oxidized with periodate to form alpha-hydroxy aldehyde groups which cross-link with the free amino groups of the biotin hydrazide. The reaction generally proceeds as follows:
  • Biotin hydrazide conjugation may be achieved using the method of D. O. Shannessy et al., Immunol. Letters 8: 243-277 (1984).
  • concentration of the antigen is adjusted to about 1.0-2.0 mg/ml in 0.1M sodium acetate, pH 5.5.
  • 0.01M sodium periodate is added to the antigen solution and the mixture is incubated in a bath at 0°C for 20 minutes.
  • Glycerol, 0.015M at 0°C is added for 5 minutes to stop the reaction.
  • the solution is then dialyzed overnight at room temperature against sodium acetate, pH 5.5.
  • Biotin hydrazide is added to a concentration of 0.01M and then the solution is incubated with gentle shaking for two hours at room temperature. The solution is then dialyzed against phosphate buffered saline, 0.01M, pH 7.4, overnight at room temperature. The biotinylated antigen is then recovered and stored at 4°C.
  • the attachment of a ligand specific for an analyte of interest to the particles of the suspension is achieved by coating the particles of the suspension with avidin, preparing a biotinylated ligand and reacting a mixture of the two to produce a suspension where the ligand is attached to the surfaces of the particles through an avidin-biotin link. Since avidin has four binding sites, such a method produces particles having a multiplicity of ligand molecules attached. The resulting particle is useful as a reagent for detecting the presence of an analyte of interest.
  • Another system contemplated comprises particles in suspension which have a biotin coating, a biotinylated ligand bound to the particle surfaces through avidin which bridges the biotin on the particle surfaces and the biotin being conjugated with the ligand.
  • Suspensions for carrying out the agglutination test of the present invention include the so-called latex suspensions which are suspensions of inert plastic particles or beads carried in a liquid vehicle, such as water.
  • the beads may be of various plastic compositions, such as a polyacrylic resin, a polystyrene resin or a polycarbonate resin. In the usual instance, the beads or particles will have diameters within the range of 0.5 microns to 3.0 microns.
  • Other contemplated suspensions are suspensions of inert particles, such as inorganic clays, as exemplified by bentonite suspensions, and fixed or tanned red blood cells.
  • a bentonite suspension may be prepared as a suspension of 0.5 g of bentonite in 500 ml distilled water and then centrifuged at 150 ⁇ g for 10 minutes. The sediment is discarded and the supernatant recentrifuged at 1400 ⁇ g for 10 minutes. This sediment is resuspended in 100 ml PBS (pH 7.4) solution in a 250-ml Erlenmeyer flask, autoclaved, and refrigerated as "stock" bentonite. Immediately before coating them with avidin, the particles are collected from a 10-ml suspension of stock bentonite and centrifuged at 1400 x g for 10 minutes.
  • the supernatant is discarded and the bentonite sediment resuspended in 2 to 3 ml 0.05 M PBS that contains avidin (1 mg/ml).
  • the avidin bentonite suspension is permitted to stand at room temperature with occasional mixing on a Vortex stirrer over a period of 30 minutes.
  • the coated particles are sedimented by centrifugation of the suspension at 1500 x g for 15 minutes and then resuspended in PBS (pH 7.4).
  • Biotinylated ligand may then be added to coat particles with the ligand and the resulting particles used in the same manner as the latex beads in an agglutination assay.
  • Tanned red cells may be coated with ligand by the following method.
  • Tanned erythrocytes are prepared from sheep blood collected in sterile 3.8 sodium citrate or Alsever solution. Blood is aged for 1 to 8 weeks at 4°C and then washed three times with PBS (pH 7.2). During the washing procedure, the buffy coat is removed by aspiration, and the remaining packed erythrocytes are resuspended to make a 2.5% concentration.
  • the optimal tannic acid (reagent grade) dilution may vary from 1:20,000 to 1:160,000 (wt/vol) and should be determined for each batch of cells. The tannic acid is diluted in PBS (pH 7.2) within one hour of use.
  • Equal volumes of the 2.5% erythrocyte suspension and the tannic acid solution are mixed and incubated in 37°C water bath for 20 minutes.
  • the cells are centrifuged at 500 x g for 10 minutes and washed once with PBS (pH 7.2). They are then resuspended to a 2.5% concentration in PBS (pH 6.7).
  • the avidin is diluted in PBS (pH 6.7) and 1 volume of the optimal avidin dilution (0.1 mg/ml) and 1 volume of 2.5% tanned cells are mixed and incubated for 30 minutes at 37°C.
  • the cells are centrifuged at 500 x g for 10 minutes, washed twice with a 1% normal swine serum in PBS (pH 6.7) diluent.
  • Biotinylated ligand prepared as previously described, may then be used to coat the red cells. The coated red cells are washed and then used in the same manner as the latex beads in an agglutination assay.
  • An agglutination test according to the present invention is conveniently carried out as in a standard slide agglutination assay.
  • a reagent comprising a suspension of particles of the present invention is mixed on a slide with a deposit of diluted or undiluted serum specimen. Mixing is accomplished by hand-tilting or mechanical rotation of the slide.
  • the latex suspension should not be permitted to dry, and if necessary, the slide may be kept covered to prevent evaporation. Reaction times may vary but, in the usual instance, are relatively short, i.e., under ten minutes.
  • the particles of the suspension visibly agglutinate.
  • a negative test the mixture on the slide does not agglutinate and appears smooth and milky.
  • the suspension was prepared by incubating a mixture of polycarbonate beads suspended in TRIS (2-amino-2-hydroxymethyl -1, 3-propanediol) buffered aqueous saline solution (pH 8.6) containing 0.02 percent by weight avidin at room temperature for two hours.
  • the particle concentration in the saline solution was 5 g per 100 ml of solution.
  • the coated particles that resulted were centrifuged and resuspended in TRIS buffered saline solution (pH 7.6) to produce a suspension containing coated particles and a particle concentration of 5 g per 100 ml of solution.
  • the resulting particles had avidin bound thereto via passive adsorption, i.e. hydrophilic or electrostatic interactions. The use of passive adsorption is faster, less expensive and less likely to inactivate the binding siteu of the avidin attached to the particle than if the avidin was covalently bound to the particle.
  • the ligand, i.e., antigen, utilized was a cell lysate of herpes simplex viral particles.
  • the antigen was used as a crude preparation of viral particles.
  • the ability to use relatively impure antigen is another advantage of the present invention. Latex particles having passive attachment of the antigen directly to the latex bead, would result in an attachment that was weak and therefore the antigen would be required to be highly purified in order to optimize binding.
  • Antigen specific for herpes simplex virus antibody was biotinylated using standard procedures. Thus, a mixture of antigen and phosphate buffered saline solution was prepared (20 mg antigen per 1 ml solution). Biotin ester (biotin-N-hydroxysuccinimide) was dissolved in dimethylformamide (50 mg ester per 1 ml solvent).
  • a mixture was prepared from 0.125 ml antigen solution, 0.02 ml sodium bicarbonate (0.5 NaHCO 3 , pH 9.0), 0.04 ml water, and 0.01 ml biotin ester solution and the mixture was incubated one hour at room temperature. After incubation, 0.025 ml of 1.0 M NH 4 Cl was added to stop further reaction. The mixture was then dialyzed against phosphate buffered saline solution. The biotinylated herpes simplex antigen was then isolated. This biotinylation procedure is mild and preserves the activity of the antigen.
  • biotinylated antigen was then mixed with the latex suspension containing avidin-coated beads using equal amounts of the antigen and the latex suspension.
  • the mixture was reacted on a vibrator at room temperature for ten minutes to produce a reagent useful in an agglutination test to detect the presence of herpes simplex virus antibody in a serum sample.
  • the latex agglutination test of the present invention prov.ides a reliable method for determining the presence or absence of an antibody in the specimen or sample tested. The test is rapidly performed and carried out far more easily than is the indirect hemagglutination test with which the agglutination test of the invention was compared.
  • Example 2 The procedure used was identical to that of Example 1 except that an activated biotin ester was used, namely N-biotinyl-6-aminocaproic acid N-hydroxy succinimide ester.
  • This biocin ester is commercially prepared as ENZOTINTM (Enzo Laboratories, New York, New York) and is a long chain activated biotinyl molecule having the following structure:
  • the N-biotinyl-6-aminocaproic acid N-hydroxy succinimide ester used may be prepared according to the method of Costello, S.M. et al. (Clin. Chem. 25(9) : 1572-1580 (1979)).
  • the long chain biotinyl molecule has a spacer arm comprising seven atoms, which is the chain comprising tne six carDons and one nitrogen group. It is contemplated that a spacer arm may be prepared which has an amino terminal end attached to a chain of from two to fourteen carbon molecules by modifying the procedure of Costello et al.
  • the present invention contemplates the use of long chain biotinyl molecules which have spacer arms having from two to fourteen carbon molecules.
  • Reaction of the long chain biotinyl molecule with the antigen results in the antigen being attached to the activated long chain biotinyl molecule via an amide bond formed between the free amino groups of lysine or arginine, or any primary amino group, in the antigen and the activated biotinyl molecule.
  • the use of a long chain biotinyl molecule decreases the steric hindrance of the antigen molecules on the surface of the particle or bead and therefore provides a more sensitive assay.
  • the linker arm (N-biotinyl-6-aminocaproic acid) may also be applied to the detection of antigens in biological fluids by binding specific antibodies to biotin via the linker arm.
  • This biotin: linker arm: antibody complex may than be contacted with the avidin coated latex bead as described earlier to create a bead with active antibody.
  • Hepatitis B surface antigens Applicants have detected Hepatitis B surface antigens in serum samples in excellent agreement with other accepted methodologies.
  • Monoclonal antibody to Hepatitis B surface antigens was obtained from Hybritech, Inc.
  • Hepatitis B surface antigens formerly known as Australian antigen, have antigenic determinants which permit their classification into various sub-types.
  • One antigenic determinant common to all Hepatitis B antigens is termed A and two mutually exclusive determinants are called D and Y.
  • the monoclonal antibody used in these studies was produced against the A:D combination.
  • the antibody protein was conjugated to the N-biotinyl-6-aminocaproic acid N-hydroxy succinimide obtained from Enzo Laboratories as ENZOTIN ⁇ as previously described.
  • biotinylated monoclonal antibody was then mixed with the latex suspension containing avidin coated beads prepared as previously described. Equal amounts of ligand and latex suspensions were used. The mixture was allowed to react on a vibrator at room temperature for 10 minutes to produce a reagent usable in an agglutination test to determine the presence of Hepatitis B antigen in various sera.
  • blood serum was drawn from 10 healthy laboratory personnel known to be negative for Hepatitis B.
  • the disclosed latex agglutination test as described found all 10 to be negative.

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Abstract

Un réactif utile pour détecter un analyte d'intérêt dans un échantillon fluide comprend: a) une particle inerte; b) une pluralité de molécules d'avidine attachées de manière non covalente à la surface de la particule; et c) des molécules de biotine dérivées formant un composé avec au moins une partie des molécules d'avidine, chaque molécule de biotine dérivée ayant la structure (I), dans laquelle A est un ligand spécifique d'un analyte d'intérêt et -R- est un groupe de liaison que relie A à la biotine de manière covalente.
PCT/US1987/000245 1986-02-06 1987-02-04 Agglutination de latex a l'aide d'un systeme d'avidine/biotine WO1987004794A1 (fr)

Applications Claiming Priority (2)

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US82732586A 1986-02-06 1986-02-06
US827,325 1986-02-06

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WO1987004794A1 true WO1987004794A1 (fr) 1987-08-13

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EP (1) EP0256117A1 (fr)
AU (1) AU7032787A (fr)
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Cited By (13)

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EP0331127A1 (fr) * 1988-02-29 1989-09-06 Roche Diagnostics GmbH Procédé pour la production d'une matrice en phase solide
US4870007A (en) * 1987-12-18 1989-09-26 Eastman Kodak Company Immobilized biotinylated receptor in test device, kit and method for determining a ligand
EP0405578A2 (fr) * 1989-06-29 1991-01-02 Nippon Shoji Kabushiki Kaisha Immunoessai enzymatique pour détecter les antigènes et phase solide pour sa mise en oeuvre
EP0416730A2 (fr) * 1989-09-08 1991-03-13 Hewlett-Packard Company Préparation d'une phase solide liant spécifiquement des substances chimiques
US5268306A (en) * 1988-02-29 1993-12-07 Boehringer Mannheim Gmbh Preparation of a solid phase matrix containing a bound specific binding pair
WO1994007140A1 (fr) * 1992-09-18 1994-03-31 Gec-Marconi Limited Procede de separation
WO1994016330A1 (fr) * 1993-01-06 1994-07-21 Beckman Instruments, Inc. Procede de preparation d'un preconjugue
GB2286672A (en) * 1993-12-23 1995-08-23 Marconi Gec Ltd Tagging substances or items
WO1995032428A1 (fr) * 1994-05-23 1995-11-30 Beckman Instruments, Inc. Reactifs et procedes de dosage rapide et quantitatif d'agents pharmacologiques
EP0769490A1 (fr) * 1995-10-19 1997-04-23 Roche Diagnostics GmbH Réactifs pour l'identification et l'isolation des carbohydrates ou des récepteurs glycaniques
CN1048464C (zh) * 1993-07-08 2000-01-19 拉斯·埃里克·特里格 一种瓶夹装置
US6235488B1 (en) 1988-09-29 2001-05-22 Agilent Technologies, Inc. Surface preparation for chemical-specific binding
EP1343009A1 (fr) * 2000-12-11 2003-09-10 Iatron Laboratories, Inc. Reactifs d'analyse immunologique et procede d'analyse

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US4870007A (en) * 1987-12-18 1989-09-26 Eastman Kodak Company Immobilized biotinylated receptor in test device, kit and method for determining a ligand
US5268306A (en) * 1988-02-29 1993-12-07 Boehringer Mannheim Gmbh Preparation of a solid phase matrix containing a bound specific binding pair
WO1989008259A1 (fr) * 1988-02-29 1989-09-08 Boehringer Mannheim Gmbh Procede pour fabriquer une matrice a l'etat solide
EP0331127A1 (fr) * 1988-02-29 1989-09-06 Roche Diagnostics GmbH Procédé pour la production d'une matrice en phase solide
US6235488B1 (en) 1988-09-29 2001-05-22 Agilent Technologies, Inc. Surface preparation for chemical-specific binding
EP0620438A1 (fr) * 1989-06-29 1994-10-19 Nippon Shoji Kabushiki Kaisha Immunoessai enzymatique pour détecter les antigènes et phase solide pour sa mise en oeuvre
EP0405578A3 (en) * 1989-06-29 1991-05-29 Nippon Shoji Kabushiki Kaisha Enzyme immunoassay for antigen and solid phase used therefor
US5378608A (en) * 1989-06-29 1995-01-03 Nippon Shoji Kabushiki Kaisha Enzyme immunoassay for antigen and solid phase used therefor
EP0405578A2 (fr) * 1989-06-29 1991-01-02 Nippon Shoji Kabushiki Kaisha Immunoessai enzymatique pour détecter les antigènes et phase solide pour sa mise en oeuvre
EP0416730A2 (fr) * 1989-09-08 1991-03-13 Hewlett-Packard Company Préparation d'une phase solide liant spécifiquement des substances chimiques
EP0416730A3 (en) * 1989-09-08 1992-07-01 Hewlett-Packard Company Substrate preparation for chemical-species-specific binding
WO1994007140A1 (fr) * 1992-09-18 1994-03-31 Gec-Marconi Limited Procede de separation
WO1994016330A1 (fr) * 1993-01-06 1994-07-21 Beckman Instruments, Inc. Procede de preparation d'un preconjugue
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GB2286672A (en) * 1993-12-23 1995-08-23 Marconi Gec Ltd Tagging substances or items
US5747352A (en) * 1994-05-23 1998-05-05 Beckman Instruments, Inc. Reagents and methods for the rapid and quantitative assay of pharmacological agents
WO1995032428A1 (fr) * 1994-05-23 1995-11-30 Beckman Instruments, Inc. Reactifs et procedes de dosage rapide et quantitatif d'agents pharmacologiques
EP0769490A1 (fr) * 1995-10-19 1997-04-23 Roche Diagnostics GmbH Réactifs pour l'identification et l'isolation des carbohydrates ou des récepteurs glycaniques
US6218546B1 (en) 1995-10-19 2001-04-17 Roche Diagnostics Gmbh Reagent for the detection and isolation of carbohydrates or glycan receptors
EP1343009A1 (fr) * 2000-12-11 2003-09-10 Iatron Laboratories, Inc. Reactifs d'analyse immunologique et procede d'analyse
EP1343009A4 (fr) * 2000-12-11 2004-05-19 Iatron Lab Reactifs d'analyse immunologique et procede d'analyse

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IL81492A0 (en) 1987-09-16
EP0256117A1 (fr) 1988-02-24

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