EP1692515A1 - Konjugate und deren verwendung in nachweisverfahren - Google Patents
Konjugate und deren verwendung in nachweisverfahrenInfo
- Publication number
- EP1692515A1 EP1692515A1 EP04797726A EP04797726A EP1692515A1 EP 1692515 A1 EP1692515 A1 EP 1692515A1 EP 04797726 A EP04797726 A EP 04797726A EP 04797726 A EP04797726 A EP 04797726A EP 1692515 A1 EP1692515 A1 EP 1692515A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- analyte
- specific binding
- antibody
- binding
- conjugate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
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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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
-
- 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
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Definitions
- the invention relates to methods for the quantitative or qualitative detection of an analyte in a sample and suitable reagents therefor.
- Binding tests are frequently used to detect analytes, in which the presence, absence or amount of the analyte in a sample can be inferred from a specific binding of the analyte to be detected to analyte-specific binding partners.
- Immunoassays or methods in which oligo- or polynucleotides are hybridized are examples of binding tests.
- the so-called “heterogeneous binding tests” are characterized by one or more separation steps and / or washing steps.
- the separation can be carried out, for example, by immunoprecipitation, precipitation with substances such as polyethylene glycol or ammonium sulfate, filtration, magnetic separation, and binding to a solid phase.
- a solid phase exists made of porous and / or non-porous, usually water-insoluble material. It can have a wide variety of shapes such as: Vessel, tube, microtitration plate, sphere, microparticles, rods, strips, filter or chromatography paper, etc.
- one of the analyte-specific binding partners is usually bound to a solid phase and is used for Separation of the binding complex "analyte / analyte-specific binding partner" from the liquid phase, while the other analyte-specific binding partner bears a detectable label (eg an enzyme, a fluorescence or chemiluminescence label, etc.) for the detection of the binding complex.
- a detectable label eg an enzyme, a fluorescence or chemiluminescence label, etc.
- test batch which contains the analyte-specific binding partners, the signal-forming components and the sample, is measured after or even during the binding reaction without a further separation and / or washing step and the corresponding measurement signal is determined.
- homogeneous immunoassays see also Boguslaski & Li (1982) Applied Biochemistry and Biotechnology, 7: 401-414
- turbidimetric or nephelometric methods • where the analyte-specific binding partners used for the detection can be associated with latex particles; EMIT ® tests; CEDIA ® tests; Fluorescence polarization immunoassays; Luminescent Oxygen Channeling Immunoassays ("LOCI”, see EP-A2-0 515 194; Ullman et al. (1994) Proc. Natl. Acad. Sei., 91: 5426-5430; Ullman et al. (1996) Clinical Chemistry , 42: 1518-1526); etc.
- LOCI see EP-A2-0 515 194; Ullman et al. (1994) Proc. Natl. Acad. Sei., 91: 5426-5430; Ullman et al. (1996) Clinical Chemistry , 42: 1518-1526); etc.
- the antibody reagents are incubated together with the sample and the measurement of the signal is carried out during and / or after the incubation without separation In other words, there is no separation of the antibody-bound analyte from the free analyte or from antibodies which have not bound any analyte.
- Homogeneous and heterogeneous binding tests can also be carried out in the form of a so-called "sandwich assay".
- the analyte is bound by a solid-phase-associated analyte-specific binding partner and an analyte-specific binding partner which is associated with a component of a signal-forming system
- Sandwich immunoassays can form antibodies or antigens or haptens as the analyte-specific binding partners.
- analyte is an antibody.
- sample analyte and reagent analyte for example a "modified analyte” such as a labeled or labeled analyte, analyte section or analyte analogue
- reagent analyte for example a "modified analyte” such as a labeled or labeled analyte, analyte section or analyte analogue
- analyte-specific binding partners In many binding tests, however, great difficulties arise in the preparation of the reagents, since the binding of the analyte-specific binding partners to solid phases or particulate components of a signal-forming system (e.g. microparticles) often results in activity losses and / or changes in properties (e.g. with regard to conformation) in the analyte-specific binding partners bound in this way or stability). This is especially true if the bound analyte-specific binding partners are proteins, such as Antibodies or enzymes.
- the detection of an analyte can be carried out by using an avidin-coated solid phase as the universal solid-phase reagent, to which the biotinylated analyte-specific binding partner is bound.
- an avidin-coated solid phase as the universal solid-phase reagent, to which the biotinylated analyte-specific binding partner is bound.
- a biotinylated analyte-specific binding partner is used which, for example, can bind to a streptavidin / enzyme complex used as a universal detection reagent. In these tests, however, separation steps, such as washing steps, are a necessary part of the test procedure.
- EP-0 356 964, EP-0 349 988 and EP-0 444 561 it is therefore considered essential for the feasibility of such a homogeneous test that the analyte-specific binding partner is monovalent with respect to the universal reagent, ie that the latter is only has a binding site for the universal reagent (eg streptavidin latex particles).
- the inventors point out that the measurement signal formation is dependent on the formation of particle pairs, which each consist of a sensitizer and a chemiluminescer particle (Ullman et al. (1996) Clinical Chemistry, 42: 1518-1526).
- a disadvantage of such methods is that the number of binding sites on the corresponding analyte-specific binding partner must be precisely controlled.
- the method according to the invention solves this problem by providing universal reagents (specific binding partners X or Y, each of which is associated with a component of a signal-forming system) which, regardless of the analyte-specific reagents (analyte-specific binding partners R1 and R2), addresses the specific needs can be adjusted, and the detection of analytes with high sensitivity and accuracy.
- this invention also solves the following general problem of homogeneous binding tests, namely the opposite one Requirements for optimal differentiation and optimal sensitivity:
- concentration of the reagents should be limited so that the background signals are as low as possible, and on the other hand the reagents should be highly concentrated and highly marked in order to achieve a sufficient signal change per unit of time.
- the analyte-specific binding partners R1 and / or R2 according to the invention are characterized in that they have more than one binding site for the specific binding partner X or Y associated with components of a signal-forming system.
- Universal reagents, the components are preferably used in the process according to the invention of a signal-forming system, which can interact with one another over very short distances, for example in the form of an energy transfer.
- the binding sites of the analyte-specific binding partners for the respective universal reagents preferably consist of small molecules, e.g. B. haptens such as digoxigenin, biotin, DNP or FITC exist, which were preferably covalently bound to the analyte-specific binding partner, the analyte-specific activity or binding capacity of R1 or R2 is generally not or hardly affected.
- the binding sites can also be part of the unchanged analyte-specific binding partner.
- the analyte-specific binding partner could e.g. be a human IgG antibody that has multiple binding sites that are specifically recognized by anti-human IgG antibodies of another species.
- a preferred object of the invention is a homogeneous method for the quantitative or qualitative detection of an analyte in a.
- Sample wherein the analyte-specific binding partner R1 has specific binding sites for the specific binding partner X, which is associated with a component of a signal-forming system, and the analyte-specific binding partner R2 has specific binding sites for the specific binding partner Y, which is associated with a component of a signal-forming system, characterized in that R1 and / or R2 has more than one binding site for the specific binding partner associated with components of a signal-forming system.
- a particular advantage of the invention is that the specific binding partners X and / or Y (for example avidin, streptavidin, etc.) do not have to be saturated as described in EP-0 138 297 by adding free “specific binding sites” (for example biotin) ,
- This method according to the invention is particularly preferably a homogeneous binding test, in particular a homogeneous immunoassay.
- this homogeneous binding test can be carried out, inter alia, in the form of a sandwich assay, an indirect immunoassay or a competitive binding test.
- a “quantitative detection” the amount, concentration or activity of the analyte in the sample is measured.
- Quantitative detection also encompasses semi-quantitative methods which only record the approximate amount, concentration or activity of the analyte in the sample or can only serve to provide a relative indication of the amount, concentration or activity.
- qualifying detection is the detection of the presence or absence of the analyte or its activity in the sample at all or the indication that the amount, concentration or activity of the analyte in the sample is below or above a certain or more certain threshold values.
- analyte to be detected in the present process substance is understood. Examples of analytes are listed in EP-A2-0 515 194 on pages 8-15.
- the analyte can be a member 'of a specific binding pair.
- the analyte may have one binding site (monovalent, usually a hapten) or several binding sites (polyvalent). In immunochemical tests, such a binding site is often also referred to as an epitope.
- the analyte can be a single substance or a group of substances which have at least one common one Have binding site.
- a monovalent analyte usually has a molecular weight of approximately 100 to 2000, in particular 125 to 1000. Many oligopeptides, oligonucleotides, oligosaccharides, drugs, drugs, metaobolites, pesticides etc. are included in the term monovalent analyte.
- a polyvalent analyte has usually a molecular weight of over 2000, mostly over 10,000. Examples of polyvalent analytes are polypeptides, polysaccharides, nucleic acids, cells, cell components including chromosomes, genes, mitochondria and other cell organelles, cell membranes, etc. Often proteins are the substances to be detected.
- Such proteins may be members of a family of proteins whose members are characterized by similar structural features and / or by a similar biological function.
- protein families of analytical interest are proteins from pathogens, immunoglobulins, cytokines, enzymes, hormones, tumor markers, metabolic markers, tissue-specific antigens, histones, albumins, globulins, skieroproteins, phosphoproteins, mucins, chromoproteins, lipoproteins, nucleoproteins, receptors, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, and glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, glycoproteins, and glycoproteins , Coagulation factors, heart attack markers (e.g. myoglobin, troponin, pro-BNP, etc.), etc.
- Other analytically interesting substances are, for example, single- or double-stranded oligo- and polynucleotides
- sample also encompasses a pretreated sample which presumably contains the substance to be detected (“analyte”), possibly in a form released or amplified by carriers.
- analyte the substance to be detected
- Some samples have to be pretreated in order to make the analyte accessible for the detection method or to remove interfering sample components.
- Such pretreatment of samples may include the separation and / or lysis of cells, the precipitation, the hydrolysis or the denaturation of sample components such as proteins, the centrifugation of samples, the treatment of the sample with organic solvents such as alcohols, especially methanol; treating the sample with detergents.
- the sample is placed in another, usually aqueous, medium transferred, which should not interfere with the detection method if possible.
- the analyte can also be amplified. Amplification of nucleic acids leads, for example, to the generation of one or more copies of the nucleic acid chain to be detected. Such amplification methods are well known to the person skilled in the art, for example “polymerase chain reaction” (PCR), “ligase chain reaction” (LCR), “amplification using Q beta replicase”, “nucleic acid sequence based amplification” (NASBA), “single primer amplification” "(ASPP) and others.
- a “analyte-specific binding partner” is either to be understood as a specific binding partner that is able to bind specifically to the analyte, or a specific binding partner (for example a modified analyte) that is able to bind to another analyte-specific binding partner.
- a “modified analyte” it is usually a substance that can bind to at least one analyte-specific binding partner, but which differs from the sample analyte in that there are no or additional binding sites, for example a biotinylated analyte or an analyte that associates with a component of a signal-forming system
- a modified analyte is used, for example, in competitive tests.
- a “specific binding partner” is understood to mean a member of a specific binding pair.
- the members of a specific binding pair are two molecules, each of which has at least one structure complementary to a structure of the other molecule, the two molecules being linked via a bond
- the term molecule also includes molecular complexes such as, for example, enzymes that consist of apo and coenzyme, proteins that consist of several subunits, lipoproteins consisting of protein and lipids, etc.
- Specific binding partners can occur naturally but also, for example, substances produced by means of chemical synthesis, microbiological techniques and / or genetic engineering processes.
- specific bin can now be used using phage display libraries, synthetic peptide databases or using "recombinatorial antibody libraries" selection partner (Larrick & Fry (1991) Human Antibodies and Hybridomas, 2: 172-189).
- specific binding partner examples include: thyroxine-binding globulin, steroid-binding proteins, antibodies, antigens, haptens, enzymes, lectins, nucleic acids, repressors, oligo- and polynucleotides, protein A, protein G, avidin, streptavidih, biotin, complement component C1q, nucleic acid-binding proteins, etc.
- Specific binding pairs are, for example: antibody-antigen, antibody-hapten, digoxigen / anti-digoxigen-antibody, fluorescin / anti-fluorescein-antibody,
- nuclease nucleotide in so-called homogeneous Gensondentesten are the specific binding partner generally nucleic acid chains, which are at least partially complementary 'to sections. the nucleic acid chain to be detected.
- antibody of this invention, for example, is an immunoglobulin in the sense, IgE, IgGl, IgG 2a, IgG 2b, IgG 3, IgG 4, IgM, to understand an immunoglobulin of the class or subclass of IgA, IgD.
- An antibody has at least one binding site (often called a paratope) for an epitope (often also called an antigenic determinant) on an antigen or hapten, which is characterized, for example, by its spatial structure and / or by the presence of polar and / or apolar groups.
- the binding site of the antibody is complementary to the epitope.
- the antigen-antibody reaction or the hapten-antibody reaction works according to the so-called “key-keyhole principle”, and is usually to a high degree specific, ie the antibodies are capable to distinguish small deviations in the primary structure, in the charge, in the spatial configuration and in the steric arrangement of the antigen or hapten.
- the so-called “complementarity determining regions" of the antibody contribute to the binding of the antibody to the antigen or hapten.
- the term "antigens" includes monovalent and polyvalent antigens.
- a polyvalent antigen is a molecule or a complex of molecules to which more than one immunoglobulin can bind at the same time, while with a monovalent antigen only one antibody can bind at a time.
- a hapten is usually a molecule that is not immunogenic by itself, - II -
- antibody is not only to be understood to mean complete antibodies but also expressly antibody fragments, such as e.g.
- Antibody fragments can be broken down, for example, by enzymatic cleavage of
- Antibody aggregates, polymers and conjugates can be generated by a variety of methods, e.g. by heat treatment, reaction with substances such as glutaraldehyde, reaction with immunoglobulin-binding molecules, biotinylation of antibodies and subsequent reaction with streptavidin or avidin, etc.
- An antibody in the sense of this invention can be a monoclonal or a polyclonal antibody.
- the antibody can be prepared by the usual methods, e.g. by immunization of humans or animals, e.g. Mouse, rat, meerschleich, rabbit, camel, horse, sheep, goat, chicken (see also Messerschmid (1996) BlOforum, 11: 500-502), and then collecting the antiserum; or by establishing hybridoma cells and then purifying the secreted antibodies; or by cloning and expression of the nucleotide sequences or modified versions thereof, which encode the amino acid sequences which are responsible for the binding of the natural antibody to the antigen and / or hapten.
- Antibodies can also be produced, if necessary with the help of genetic engineering methods in plants - such as Yeast cells - (Fischer et al. (1999) Biol. Chem., 380: 825-839; Hiatt et al. (1992) Genetic Engineering, 14: 49-64)), animal and prokaryotic cells (see for example WO 95/25172) as well as isolated human cells.
- plants - such as Yeast cells - (Fischer et al. (1999) Biol. Chem., 380: 825-839; Hiatt et al. (1992) Genetic Engineering, 14: 49-64)
- animal and prokaryotic cells see for example WO 95/25172 as well as isolated human cells.
- a “signal-forming system” can be one or more components, at least one component being one verifiable label.
- a label is any molecule that can itself produce a signal or can induce the production of a signal, such as a fluorescent substance, a radioactive substance, an enzyme, or a chemiluminescent substance.
- the signal can be detected or measured, for example, on the basis of the enzyme activity, the luminescence, the light absorption, the light scattering, the emitted electromagnetic or radioactive radiation, or a chemical reaction.
- a “label” is capable of generating a detectable signal itself, so that no further components are necessary. Many organic molecules absorb ultraviolet and visible light, whereby the energy transmitted by the light absorption can bring these molecules into an excited energy state, and the absorbed energy in the form emit light of a different wavelength than that of the incident light, and yet other labels can directly generate a detectable signal, such as radioactive isotopes, dyes or magnetic and non-magnetic microparticles.
- the signal-producing system includes all the components required for signal generation, such as Substrates, coenzymes, quenchers, accelerators, additional enzymes, substances that react with enzyme products, catalysts, activators, cofactors, inhibitors, ions etc.
- Suitable labels are, for example, enzymes including horseradish peroxidase, alkaline phosphatase, glucose-6 Phosphate dehydrogenase, alcohol dehydrogenase, glucose oxidase, ⁇ -galactosidase, luciferase, urease and acetylcholinesterase; dyes; fluorescent substances including fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, ethidium bromide, 5-dimethylaminonapthalene-1-sulfonyl chloride and fluorescent rare earth chelates; chemiluminescent substances including luminol, isoluminol, acridinium compounds, olefin, enol ether
- a signal-generating system can also comprise components which can interact with one another in the vicinity of one another, for example in the form of energy donors and energy receivers such as photosensitizers and chemiluminescent substances (EP-A2-0 515 194), photosensitizers and fluorophores (WO 95 / 06877), radioactive iodine 125 and fluorophores (Udenfriend et al. (1985) Proc. Natl. Acad. Sci. 82: 8672-8676), fluorophores and fluorophores (Mathis (1993) Clin. Chem. 39: 1953-1959) or Fluorophores and fluorescence quencher (US 3,996,345).
- photosensitizers and chemiluminescent substances EP-A2-0 515 194
- photosensitizers and fluorophores WO 95 / 06877
- radioactive iodine 125 and fluorophores Udenfriend et al. (1985) Proc. Natl. Acad
- Interaction between the components is the direct transfer of energy between the components, e.g. by light or electron radiation as well as via short-lived reactive chemical molecules, - included.
- This also includes processes in which the activity of a component is inhibited or enhanced by one or more others, for example the inhibition or increase in the enzyme activity or the inhibition, increase or change (for example wavelength shift, polarization) of the electromagnetic component emitted by the affected component Radiation.
- the interaction between the components also includes enzyme cascades.
- the components are enzymes, at least one of which supplies the substrate for another, so that a maximum or minimum reaction rate of the coupled substrate conversion results.
- An effective interaction between the components usually takes place when they are spatially adjacent, that is, for example, within a distance range of a few ⁇ m, in particular within a distance range of less than 600 nm, preferably less than 400 nm, very particularly preferably less than 200 nm.
- the signal-forming system comprises photosensitizers associated with microparticles and chemiluminescent substances associated with microparticles.
- Microparticles are often used as a solid phase and / or as a label.
- the term “microparticles” is understood to mean particles which have an approximate diameter of at least 20 nm and not more than 20 ⁇ m, usually between 40 nm and 10 ⁇ m, preferably between 0.1 and 10 ⁇ m, particularly preferably between 0.1 and 5 ⁇ m, very particularly preferably between 0.15 and 2 ⁇ m.
- the microparticles can have a regular or irregular shape. They can be spheres, spheroids, spheres with more or less large cavities or pores.
- the microparticles can be organic , of inorganic material or of a mixture or a combination of both.
- microparticles can consist of a porous or non-porous, a swellable or non-swellable material.
- the microparticles can have any density, but particles with a density that is preferred are preferred approximates to about 0.7 to about 1.5 g / ml of water.
- the preferred microparticles are in water aqueous solutions can be suspended and suspension stable for as long as possible. They may be translucent, partially translucent, or opaque.
- the microparticles can consist of several layers, such as the so-called “core-and-shell” particles with a core and one or more enveloping layers.
- microparticles includes, for example, dye crystals, metal sols, silica particles, glass particles, magnetic particles, polymer particles, oil drops, Lipid particles, dextran and protein aggregates.
- Preferred microparticles are particles which can be suspended in aqueous solutions and which consist of water-insoluble polymer material, in particular of substituted polyethylenes.
- Latex particles for example made of polystyrene, acrylic acid polymers, methacrylic acid polymers, acrylonitrile polymers, acrylonitrile butadiene styrene, polyvinyl acetate, are very particularly preferred Acrylate, polyvinylpyridine, vinyl chloride-acrylate.
- latex particles with reactive groups on their surface such as carboxyl, amino or Aldehyde groups that allow covalent binding, for example, of specific binding partners to the latex particles.
- reactive groups on their surface such as carboxyl, amino or Aldehyde groups that allow covalent binding, for example, of specific binding partners to the latex particles.
- the production of latex particles is described for example in EP 0 080 614, EP 0 227 054 and EP 0 246 446.
- association is to be understood broadly and includes, for example, a covalent and a non-covalent bond, a direct and an indirect bond, the adsorption to a surface and the inclusion in a depression or a cavity, etc.
- a covalent bond For example, the specific binding partner is bound to a label by means of a chemical bond.
- a covalent bond between two molecules if at least one atomic nucleus of one molecule shares electrons with at least one atomic nucleus of the second molecule.
- a non-covalent bond examples include surface adsorption, inclusion in cavities or the binding of two specific binding partners
- the specific binding partners can also be indirectly bound to the label via specific interaction with other specific binding partners illustrated:
- a biotinylated anti-fluorescein antibody can be bound to the label via label-bound avidin.
- a microparticle can have a coating of one or more layers, e.g. from proteins, carbohydrates, biopolymers, organic polymers or mixtures thereof, for example to suppress or prevent the non-specific binding of sample components to the particle surface, or to achieve improvements in terms of suspension stability, storage stability, shaping stability or resistance to UV radiation, for example Light, microbes or other destructive agents.
- this coating can in particular from protein layers or polymer layers, such as. B. cyclodextrins, dextrans, hydrogels, albumin or polyalbumin exist, which have been applied covalently or adsorptively on the microparticles.
- R1 and / or R2 can be bound to components of the signal-forming system before, during or after the binding reaction with the analyte via X and / or Y.
- the sample can first be incubated with the analyte-specific binding partners R1 and R2 and then the specific binding partners X and Y are added.
- a different order of reagent addition can also take place.
- the sample is first mixed with the analyte-specific binding partners R1 and R2 in a sequential or simultaneous sequence and then the components of the signal-forming system with the binding partners X and Y are added to the mixture in a sequential or simultaneous sequence ,
- the number of binding sites of the analyte-specific binding partner R1 according to the invention for the specific binding partner X should be at least 2, preferably at least 5, particularly preferably at least 10 and very particularly preferably at least 15 and the number of binding sites of the analyte-specific binding partner R2 according to the invention for the specific binding partner Y should be at least 2, preferably at least 5, particularly preferably at least 10 and very particularly preferably at least 15.
- the analyte-specific binding partners R1 and R2 according to the invention can also be the same or different analyte-specific binding partners.
- a monoclonal antibody can be used both as analyte-specific binding partner R1 and as analyte-specific binding partner R2 if the analyte has more than one epitope for this antibody.
- the analyte-specific binding partners R1 and R2 can both bind the analyte specifically.
- the analyte-specific binding partners can, for example, be analyte-specific antibodies in the case of a sandwich immunoassay or, if the analyte itself is an antibody, its antigen or a "modified antigen" or an antigen analogue. It should be a competitive test setup one of the analyte-specific binding partners R1 and R2 according to the invention is a modified analyte. Both .
- Binding sites of the analyte-specific binding partner R1 according to the invention for the specific binding partner X are preferably haptens.
- Particularly preferred as binding sites of the analyte-specific binding partner R1 according to the invention are biotin, digoxigenin, fluorescein, single-stranded nucleic acid chains, dinitrophenol.
- other molecules that are a member of a specific binding pair can also be used.
- binding sites of the analyte-specific binding partner R2 according to the invention for the specific binding partner Y are preferably haptens.
- Particularly preferred as binding sites of the analyte-specific binding partner R2 according to the invention are biotin, digoxigenin, fluorescein, single-stranded nucleic acid chains, dinitrophenol.
- other molecules that are a member of a specific binding pair can also be used.
- components of the signal-forming system are brought into a distance from one another which creates an interaction, in particular an energy transfer, between them Components allowed. The extent of this interaction is then measured for quantitative or qualitative detection of the analyte in the sample.
- the method is particularly suitable for sandwich assays and indirect immunoassays.
- components of the signal-forming system are brought into a distance from one another which has no or only a very slight interaction, in particular no or only very little energy transfer between them Components allowed. The remaining extent of this interaction is then measured for quantitative or qualitative detection of the analyte in the sample.
- This method is particularly suitable for competitive binding tests.
- Carrier molecules are introduced to which both the analyte-specific
- Binding partner as well as the binding sites can be bound. It is therefore advantageous in the method according to the invention if R1 is one or more analyte-specific binding partners which are associated with a carrier molecule, the carrier molecule being binding sites for the specific one
- Binding partner X can have. It is also advantageous if it is also
- R2 is one or more analyte-specific binding partners that deal with a
- Carrier molecule are associated, wherein the carrier molecule can have binding sites for the specific binding partner Y.
- R1 and / or R2 are each associated with such a carrier molecule.
- Suitable carrier molecules are, for example, proteins, for example antibodies, enzymes, albumins such as bovine serum albumin or human serum albumin, or protein polymers, dextrans, cyclodextrins, dendrimers or similar structures.
- Particularly preferred protein polymers or aggregates can consist of antibodies, albumin molecules, enzymes or mixtures thereof which are associated with one another, preferably covalently bound.
- biotinylated dextran and biotinylated protein polymers eg biotinylated antibody polymers).
- a particularly preferred carrier molecule can be prepared as described in Example 7.
- murine antibodies preferably murine IgG antibodies
- carrier molecules according to the invention can also be produced from enzymes, antibodies (e.g. antibodies, in particular IgG antibodies, from the mouse or the goat), albumins or mixtures thereof.
- the binding sites for the specific binding partner X or Y e.g. Biotin, digoxigenin, fluorescein, single-stranded nucleic acid chains, dinitrophenol, etc. can be bound to these protein polymers using methods known to those skilled in the art, covalent binding being preferred.
- the binding sites for the specific binding partner X or Y can be introduced before or as described in Example 7 after the binding reaction between analyte-specific binding partner and carrier molecule.
- analyte-specific binding partner can be bound to a carrier molecule.
- several carrier molecules can also be bound to one analyte-specific binding partner.
- Another object of this invention is a carrier molecule which is associated with one or more analyte-specific binding partners, preferably covalently linked.
- the carrier molecule is a protein polymer, for example covalently linked to one another Antibodies (see Example 7), albumin molecules, enzymes or mixtures thereof, which may additionally have binding sites, for example biotin, digoxigenin, fluorescein, single-stranded nucleic acid chains, dinitrophenol, etc., for a specific binding partner X or Y.
- the number of binding sites should be at least 2, better more than 5, preferably more than 10, particularly preferably more than 15, very particularly preferably more than 18 per carrier molecule which is associated with one or more analyte-specific binding partners.
- the analyte-specific binding partner in this embodiment according to the invention is preferably an antibody or an antibody fragment, an antigen, a hapten or a nucleic acid chain.
- Analyte-specific binding partners which are associated with the carrier molecules according to the invention are also called conjugate below. These can of course not only be used in homogeneous binding tests but also advantageously in heterogeneous binding tests.
- a conjugate according to the invention consists of a carrier molecule which is associated with one or more analyte-specific binding partners, this conjugate having additional binding sites for a specific binding partner X or Y.
- the carrier molecule consists of dextran, cyclodextrin, dendrimers or of linked antibodies, linked albumin molecules, linked enzymes or linked mixtures thereof. Their binding should preferably be covalent.
- the additional binding sites of the conjugate according to the invention can be biotin, digoxigenin, fluorescein, dinitrophenol or single-stranded nucleic acid chains.
- the carrier molecule is covalent to one or more analyte-specific Binding partner bound.
- the conjugate according to the invention should have at least 2, preferably more than 5, particularly preferably more than 10, very particularly preferably more than 15 and optimally more than 18 additional binding sites for the specific binding partner X or Y.
- a conjugate in which the carrier molecule consists of antibodies covalently linked to one another, preferably IgG antibodies from the mouse or the goat, is very particularly preferred.
- Another embodiment of the invention is a reagent which contains one or more of these conjugates and a test kit which contains such a reagent.
- the conjugates according to the invention can be used in a homogeneous or heterogeneous binding test (for example an immunoassay) for the quantitative or qualitative detection of an analyte in a sample.
- a binding test according to the invention in particular a homogeneous binding test, a conjugate according to the invention which has specific binding sites for the specific binding partner X associated with a component of a signal-forming system is used for the quantitative or qualitative detection of an analyte in a sample.
- a further conjugate according to the invention which has specific binding sites for the specific binding partner Y associated with a component of a signal-forming system, is additionally used for the quantitative or qualitative detection of an analyte in a sample.
- the number of binding sites for the specific binding partner X or Y should be at least 2, preferably at least 5, particularly preferably at least 10 and very particularly preferably at least 15.
- X and Y can be the same or a different specific binding partner.
- Preferred binding partners X and Y are avidin, streptavidin, anti-digoxigenin antibodies, anti-dinitrophenol antibodies, single-stranded nucleic acid chains, anti-hapten antibodies, enzymes, enzyme substrates or antibodies, which contain specific polypeptides, oligopeptides or enzymes bind, used.
- a binding test preferably a homogeneous binding test, using one or more of the conjugates according to the invention is very particularly preferred within the meaning of this invention, components of the signal-forming system being brought into a distance from one another as a result of the binding of the analyte-specific binding partners, which interaction, in particular one Energy transfer between these components is permitted and the extent of this interaction is measured or, as a result of the binding of the analyte-specific binding partners, components of the signal-forming system are brought into a distance from one another which has no or only very little interaction, in particular no or only very little energy transfer, between them Components allowed and the remaining extent of this interaction is measured.
- microparticles in particular latex particles, are preferably used as components of the signal-forming system.
- photosensitizers associated with microparticles and chemiluminescent substances associated with microparticles are very particularly preferably used as components of the signal-forming system.
- microparticles in particular latex particles
- Sensitizers associated with microparticles in particular photosensitizers, and chemiluminescent substances associated with microparticles are very particularly preferred as components of the signal-forming system.
- a microparticle is a further subject of this invention, in particular a latex particle, to which a conjugate according to the invention is bound via a specific binding partner X or Y bound to the microparticle and its use in a method according to the invention.
- a microparticle which is associated as a component of a signal-forming system with photosensitizers or with chemiluminescent substances, is preferred.
- a particularly preferred embodiment of the method according to the invention is a test based on the LOCI method, which is described in detail in EP-0 515 194. This is based on the use of photosensitizers and so-called acceptors as signal-generating components.
- the photosensitizers generate exposure for singlet oxygen with the acceptors, the chemiluminescent components, • responding.
- the activated chemiluminescent component produces light, which is measured.
- the analyte is bound, for example, to an analyte-specific binding partner R1, which may be associated with a carrier molecule, which is bound to so-called sensitizer particles by means of the specific binding partner X.
- the sensitizer molecules associated with the sensitizer particle can generate singlet oxygen in the excited state. This singlet oxygen can react with the chemiluminescent compounds, which are associated with so-called chemiluminescent particles, the metastable compound formed again disintegrating to produce a flash of light.
- the analyte-specific binding partner R2 which can be associated with a carrier molecule, is bound to the chemiluminescer particles by means of the specific binding partner Y. Since singlet oxygen is only stable for a short time in aqueous solutions, ⁇ the chemiluminescer particles associated with analyte-specific binding partners, which have come into close proximity to the sensitizer particles excited by light, for example, are stimulated to emit light. The wavelength of the emitted light to be measured can be changed by appropriate fluorescent dyes in the chemiluminescer particles. In this method, the sample is preferably first incubated with the analyte-specific binding partners R1 and R2 and then the specific binding partners X and Y, which are associated with the sensitizer or chemiluminescer particles, are added.
- the analyte-specific binding partner R1 is associated with a component of a signal-forming system and the analyte-specific binding partner R2 has specific binding sites for the specific binding partner Y, which is associated with a component of a signal-forming system, R2 being more than one Binding site for the respective specific binding partner Y associated with components of a signal-forming system.
- a reagent in liquid or lyophilized form is an object of this invention which contains one or more of the carrier molecules according to the invention described above, each of which is associated with one or more analyte-specific binding partners, or which contains the microparticles according to the invention.
- a test kit containing such a reagent is also encompassed by this invention. This also applies to the use of this reagent and / or the test kit for carrying out a homogeneous or heterogeneous binding test for the quantitative or qualitative detection of an analyte in a sample, preferably for carrying out one of the homogeneous methods described in the patent claims.
- test kit for performing the homogeneous binding test according to the invention.
- This test kit is characterized in that it contains an analyte-specific binding partner R1, which has more than one specific binding site for the specific binding partner X, which is associated with a component of a signal-forming system, and in that it contains an analyte-specific binding partner R2, which has more than one has specific binding site for the specific binding partner Y, which is associated with a component of a signal-forming system.
- test kit according to the invention can also additionally contain the specific binding partners X and / or Y associated with components of a signal-forming system. Furthermore, the test kits according to the invention can also contain a package insert, dilution buffer, standards, controls, system reagents and / or further reagents and materials (e.g. cuvettes, sampling devices) required for carrying out the test.
- a package insert e.g. dilution buffer, standards, controls, system reagents and / or further reagents and materials (e.g. cuvettes, sampling devices) required for carrying out the test.
- test kits contain an analyte-specific binding partner R1, which consists of one or more analyte-specific binding partners with biotinylated dextran, biotinylated protein polymers, biotinylated antibody polymer or an are associated with another carrier molecule and / or an analyte-specific binding partner R2, which consists of one or more analyte-specific binding partners which are associated with biotinylated dextran, biotinylated protein polymers, biotinylated antibody polymers or another carrier molecule.
- R1 an analyte-specific binding partner
- R2 which consists of one or more analyte-specific binding partners with biotinylated dextran, biotinylated protein polymers, biotinylated antibody polymers or another carrier molecule.
- a solution of chlorophyll-a in benzyl alcohol (1.0 ml; 0.6 mM) is added to 8.0 ml of benzyl alcohol which has been heated to 105 ° C.
- a suspension of latex beads (175 nm, carboxyl-modified latex, Bangs Laboratories, Carmel, IN) in water (10%; 1.0 ml) is added to the benzyl alcohol solution.
- the mixture is stirred at 105 ° C for 5 minutes and then cooled to room temperature.
- 10 ml of ethanol are added and the mixture is centrifuged.
- the pellet is resuspended in a 1: 1 water-ethanol mixture (10 ml) and then centrifuged again. The same procedure is repeated with water and the pellet is then taken up in physiological saline solution.
- chemiluminescent particles 20 ml of the carboxyl-modified latex particle suspension (10% suspension in water) are mixed with 20 ml of 2-ethoxyethanol. The mixture is heated to 90 ° C. 20 ml of a solution of 10 mM dioxen, 20 mM europium chelate with the agent 3- (2-thienoyl) -1, 1, 1trifluoroacetone (Kodak, CAS # 14054-87-6) (EuTTA) and 60 mM trioctylphosphine oxide (TOPO) in 2-Ethoxyethanol are added to the suspension of particles. The mixture is further heated at 97 ° C for 7 minutes.
- the coupling conditions during the incubation 50 mg particles / ml coupling, 0.5 mg sodium cyanoborohydride / ml coupling, 2 mg streptavidin / 20 mg particles.
- the supernatant was separated by centrifugation and the particles were resuspended in coupling buffer (with 0.6 M NaCl, Merck). The supernatant was then separated off again by centrifugation and the particles in storage buffer (0.1 M Tris HCl, 0.3 M NaCl, 25 mM EDTA, 0.1% BSA, 0.1% Dextran T-500, 0.1% Zwittergent 3-14, 0.01% gentamycin, 15 ppm ProClin-300, pH 8.0) and resuspended.
- Sensitizer particles with anti-digoxioenin 15 mg of sensitizer particles with 3 mg anti-digoxigenin antibody (Mab DIG 2H6, Dade Behring Inc.) and 0.15 mg sodium cyanoborohydride (Sigma, S 8628) were obtained in a coupling buffer (0.05 M ß-morpholino-ethanesulfonic acid; Serva, Art. 29834) combined and incubated for 24 hours at + 37 ° C. The coupling conditions during the incubation: 32.8 mg particles / ml coupling, 6.6 mg antibody / ml coupling, 0.33 mg sodium cyanoborohydride / ml coupling, 3 mg antibody / 15 mg particles.
- a coupling buffer 0.05 M ß-morpholino-ethanesulfonic acid
- the supernatant was separated by centrifugation and the particles were resuspended in coupling buffer (with 0.6 M NaCl, Merck). The supernatant was then separated off again by centrifugation and the particles in storage buffer (0.1 M Tris HCl, 0.3 M NaCl, 25 mM EDTA, 0.1% BSA, 0.1% Dextran T-500, 0.1% Zwittergent 3-14, 0.01% gentamycin, 15ppm ProClin-300, pH 8.0) and resuspended.
- coupling buffer with 0.6 M NaCl, Merck.
- storage buffer 0.1 M Tris HCl, 0.3 M NaCl, 25 mM EDTA, 0.1% BSA, 0.1% Dextran T-500, 0.1% Zwittergent 3-14, 0.01% gentamycin, 15ppm ProClin-300, pH 8.0
- Chemiluminescencer particles acceptor particles
- anti-digoxigenin or anti-troponin Aceeptor particles were coupled analogously to the coupling "Sensitizer particles with anti-digoxigenin” with antibodies directed against digoxigenin or troponin.
- Universal reagent A (Aceeptor particles with streptavidin) is used in combination with Universal reagent B (Sensitizer particles with anti-digoxigenin) or Universal reagent A (Aceeptor particles with anti-digoxigenin) is used in combination with Universal reagent B (Sensitizer particles with streptavidin) used the following examples.
- Conjugate K1 biotinylated anti-PSA antibody
- Conjugate K2 (anti-PSA antibody labeled with digoxigenin): The anti-PSA antibody (MAK ⁇ PSA> 92-283 / 029 Dade Behring Marburg GmbH) was treated with digoxigenin according to the instructions / instructions for use DIG-Antibody Labeling Kit (Boehringer Mannheim Biochemica, order no. 1367200, implementation: protein labeling with DIG-NHS LMonoclonal antibodies).
- the anti-PSA antibody (MAK ⁇ PSA> 92-284 / 03, Dade Behring Marburg GmbH) was used different amounts of biofin molecules and the anti-PSA antibody (MAK ⁇ PSA> 92-283 / 029, Dade Behring Marburg GmbH) conjugated with different amounts of digoxigenin molecules.
- the results of the antibody pairs are shown in the following table.
- Table 3 PSA assay based on antibodies, with a different number of binding sites for the specific binding partners X and Y. Measurement signal given in "Counts”.
- the conjugate is then desalted over a PD-10 column with phosphate buffer 0.1 M pH 6.0.
- phosphate buffer 0.1 M pH 6.0 Manufacture of activated biotin dextran
- 1.7 mL of the antibody-SATA solution (1 mg / mL) are mixed with 509 ⁇ L of the FlukaBioDex-GMBS solution (1.32 mg / mL). This mixture is incubated for 2 hours at 37 ° C. and then stopped with 220 ⁇ L 0.1 M n-ethylmaleimide solution.
- the purification / desalination is carried out using Sephacryl S300 (diameter 1.6 cm, gel bed height 90 cm, application amount approx. 2 mL) with 0.1 M TRIS / HCl, 150 mM NaCI pH 7.4.
- the fractions containing the conjugate were pooled and concentrated to 1.7 mL ( «0.65 mg / mL).
- 1.7 mL of the antibody SATA solution (1 mg / mL) are mixed with 102 ⁇ L of the FlukaBioDex-GMBS solution (1.32 mg / mL). This mixture is incubated for 2 hours at 37 ° C. and then stopped with 180 ⁇ L 0.1 M n-ethylmaleimide solution.
- the purification / desalination is carried out using Sephacryl S300 from Fluka (diameter 1.6 cm, gel bed height 90 cm, application amount approx. 2 mL) with 0.1 M TRIS / HCl, 150 mM NaCI pH 7.4.
- the fractions containing the conjugate were pooled and concentrated to 1.7 mL ( «0.64 mg / mL).
- Table 4 PSA assay using dextran-antibody-biotin conjugates. Measurement signal specified in "Counts”.
- Conjugate K1 biotinylated anti-human IgM antibody
- the anti-rubella antibody (MAK ⁇ Rubella> 93-9 / 08, Dade Behring Marburg GmbH) was digoxigenin according to the instructions / instructions for use DIG-Antibody Labeling Kit (from Boehringer Mannheim Biochemica, order no. 1367200, implementation: protein -Labeled with DIG-NHS LMonoclonal Antibodies).
- Example 7 Comparison of a standard biotin conjugate with a biotinylated carrier molecule Fab 'conjugate using a troponin assay
- M-IgG murine IgG antibodies
- 0.1 M phosphate buffer 5 mM EDTA, pH 7.0
- an aqueous sulfosuccinimidyl (4-iodoacetyl) aminobenzoate solution (0.66 mL, 2.0 mg / mL).
- the mixture is concentrated and purified using a gel filtration column (AcA22, Ciphergen, Fremont, CA). The fractions with the monomeric, activated M-IgG (tested with HPLC) are pooled.
- a solution (3.0 mL, 10 mg / mL) of an F (ab ') 2 fragment of an anti-troponin antibody in a 0.1 M phosphate buffer (5 mM EDTA, pH 6.0) is mixed with 0.091 mL of a mixture of dithiothreitol ( 15.4 mg / mL) and 2-mercaptoethanol (15.6 uL / mL) mixed.
- the mixture is concentrated and purified using a gel filtration column (AcA22, Ciphergen, Fremont, CA). The fractions with the Fab 'antibody fragment are pooled.
- the Fab '-M-IgG conjugate in the phosphate buffer pH 7.0 (6 mL; 0.8 mg / mL; 16 ⁇ mol) is mixed with an aqueous solution of the NHS-PEO4-Biotin (Pieree Chemical Company, Rockford, ILL; 0.095 mL, 0.5 mg / mL). After a reaction time of 4 hours at room temperature, the mixture is diafiltered against the pH 7.0 phosphate buffer.
Abstract
Description
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US52605403P | 2003-12-01 | 2003-12-01 | |
US10/725,166 US20050118727A1 (en) | 2003-12-01 | 2003-12-01 | Conjugates and their use in detection methods |
PCT/EP2004/012647 WO2005062057A1 (de) | 2003-12-01 | 2004-11-09 | Konjugate und deren verwendung in nachweisverfahren |
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US20090220989A1 (en) * | 2005-12-05 | 2009-09-03 | Guava Technologies | Particle-Based Analyte Characterization |
DE102007043281A1 (de) | 2007-09-11 | 2009-05-28 | Sebastian Dr. med. Chakrit Bhakdi | Vorrichtung, Materialien und Verfahren zur Hochgradientenmagnetseparation biologischen Materials |
CA3016361C (en) | 2016-03-07 | 2024-01-02 | X-Zell Inc. | Compositions and methods for identifying rare cells |
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US4434150A (en) * | 1981-10-19 | 1984-02-28 | Ortho Diagnostic Systems, Inc. | Immunological reagents employing polymeric backbone possessing reactive functional groups |
US4778751A (en) * | 1986-05-12 | 1988-10-18 | Diagnostic Products Corporation | Method for measuring antigens or antibodies in biological fluids using ligand labeled antigens or ligand labeled antibodies |
JPH0534346A (ja) * | 1990-09-14 | 1993-02-09 | Tosoh Corp | 免疫測定法 |
DE69213240T2 (de) * | 1991-07-04 | 1997-04-24 | Immunodex K S | Wasserlösliche reagenzien und konjugate auf polymerbasis, die vom divinylsulfon abgeleitete reste enthalten |
JP3544962B2 (ja) * | 2000-08-21 | 2004-07-21 | エフ.ホフマン−ラ ロシュ アーゲー | 改良された結合アッセイ |
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DE10064827A1 (de) * | 2000-12-22 | 2002-06-27 | Dade Behring Marburg Gmbh | Nachweisverfahren |
JP2003075443A (ja) * | 2001-09-03 | 2003-03-12 | Sekisui Chem Co Ltd | 標的物質測定試薬、それを用いた標的物質の測定方法 |
JP2003194817A (ja) * | 2001-12-26 | 2003-07-09 | Techno Network Shikoku Co Ltd | イムノクロマトグラフ分析の測定方法 |
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