EP0874989A1

EP0874989A1 - Immunoassay with the aid of a mixture of detector molecules forming a detector substance

Info

Publication number: EP0874989A1
Application number: EP96933353A
Authority: EP
Inventors: Wolf-Georg Forssmann; Knut -Nieders. Inst. Peptid Fors. GMBH ADERMANN; Andreas -Nieder. Inst. Peptid Fors. GMBH KIST
Original assignee: FORSSMANN Wolf-Georg Prof Dr med; Haemopep Pharma GmbH
Current assignee: FORSSMANN Wolf-Georg Prof Dr med; Haemopep Pharma GmbH
Priority date: 1995-09-21
Filing date: 1996-09-21
Publication date: 1998-11-04
Also published as: DE19534988A1; JPH11512527A; WO1997011372A1; AU7212296A

Abstract

The disclosure pertains to an immunoassay method using a detector substance which interacts with a substrate to be detected. The detector substance is a mixture of at least two different types of detector molecule; these detector molecules each carry at different positions a chemical group A which is capable of forming an affinity bond; the detector substance comprising at least two different types of detector molecule is brought into contact with a sample containing the substrate to be detected; once complexes are formed between the substrate and the two types of detector molecule, they are immobilised and/or detected with an affinity substance which forms an affinity bond with the chemical group A.

Description

Immunoassay with a detector molecule that forms a detector substance

The present invention relates to an immunoassay with a detector substance which interacts with a substrate to be detected according to claim 1, a detector substance according to claim 6, uses of the detector substance according to claim 9 and a diagnostic agent containing the detector substance according to claim 12.

Immunometric assays, also called sandwich assays or two-site assays, work according to the double antibody principle with two antibodies that bind different epitopes of the antigen. Here, the wall antibody is immobilized in excess on a support, for example on the wall of the incubation vessel. This antibody selectively captures the antigen to be measured from the offered sample. In a separation step, all substances are removed from the sample that were not bound by the wall antibody. A second, labeled antibody (labeling, for example, radioactive, chemiluminometric, enzymatic, or fluorometric) then attaches itself to another epitope of the bound antigen. Here, the two antibodies must not interfere with one another sterically for the purpose of optimal binding to the antigen. Unbound second antibody is also separated and removed from the sample. A measurement signal (eg radioactivity, chemiluminescence, enzymatic color reaction, or fluorescence) is only generated by the complex wall-antibody-antigen-second antibody marker. For the development of an immunometric assay it is necessary to have antibodies which bind to different epitopes of the antigen. These epitopes must be at a sufficient distance from one another in order to enable the simultaneous binding of both antibodies without steric hindrance. About 12 to 16 amino acids of the antigen are involved in the binding with the antibody (Amit et al., 1986, Science 233, 747-753; Fischmann et al., 1991, J. Biol. Chem. 266, 12915-12920). In order to avoid mutual steric hindrance of the two antibodies, it is therefore necessary to generate region-specific antibodies which bind as precisely as possible to previously defined areas of the antigen. This is all the more true the smaller the antigen.

The binding between antigen and antibody can depend very much on the way in which the antigen is presented. This is especially true for antibodies that are directed against epitopes that change significantly as the milieu changes. In addition to pH and ionic strength, such changes in the environment can also be the binding to a solid phase. Epitopes that are defined in the tertiary and quaternary structure of the antigen are primarily affected.

In the classic immobilization assay, the wall antibody is adsorbed directly onto the polystyrene surface of microtiter plates. As is generally the case with peptides and proteins, the binding to the plastic surface is based on non-specific, hydrophobic interactions and is accordingly weak. The antigen can also be bound directly to the surface of the microtiter plates based on non-specific, hydrophobic interactions. The disadvantages of such a relatively weak binding, however, are the fact that the binding site on the wall surface cannot be predetermined and the high proportion of desorption during the assay procedure.

The technical problem on which the invention is based is to provide an immunoassay which is basically a ensures higher sensitivity and higher specificity. The techniques for detection that are common in immunoassay technology should remain applicable.

The technical problem is solved by the present invention. The general idea of the invention here is that the principle described does not use a single detector substance with an affinity grouping that is nonspecifically distributed on the detector substance, but rather uses mixtures of at least two detector molecules that position the affinity grouping at different but specific positions of the respective detector molecule. This advantageously significantly reduces the risk of a negative interaction of the detector molecule provided with the affinity group with the substrate to be detected and the sensitivity of such an immunoassay functioning in this way is significantly increased.

The technical problem on which the invention is based is in particular solved by an immunoassay with the features of patent claim 1. The subsequent subclaims 2 to 5 relate to preferred embodiments of the immunoassay. Patent claim 6 relates to the detector substance to be used according to the invention, which comprises a mixture of at least two detector molecules, patent claim 9 relates to the use of the detector substance and claim 12 relates to a diagnostic agent containing the detector substance according to the invention.

The immunoassay according to the invention is carried out with a detector substance which interacts with a substrate to be detected. According to the invention, the detector substance is a mixture of at least two different detector molecules. The detector molecules each have a chemical grouping A at different positions, which is suitable for entering into an affinity bond. The detector substance according to the invention therefore contains at least two classes of detector molecules, one class being the chemical grouping A at a specific position which is different from the specific position of the second class of detector molecules. The detector molecules are preferably present in approximately the same concentration in the mixture forming the detector substance. According to the invention, the detector substance comprising at least two detector molecules is brought into contact with a sample which contains the substrates to be detected. After complexes have been formed from the substrate to be detected and the two different detector molecules in each case, these are immobilized and / or detected by complexes which are formed by an affinity substance which forms an affinity bond with the chemical group A.

The detector molecule is preferably a polypeptide, in particular an antibody, which can specifically interact with an antigen, glycosylated peptides which, due to their polysaccharide-containing grouping, can specifically interact, for example lectins, oligosaccharides, nucleic acids and haptens.

Chemical group A preferably comprises biotin, streptavidin, antigen, antibody, protein A, F _c fragments, F _ab fragments, hormones, receptors, enzymes, enzyme substrates.

The affinity substance, which is able to form a complex with chemical group A, is complementary to chemical group A. Thus, for example, when using a detector molecule which carries the biotin as chemical group A, the affinity substance with the complex formed from the detector The substrate and detector molecule are immobilized or it is proven that streptavidin is. Similarly, the chemical grouping A can be, for example, protein A, whereas the affinity substance is then an antibody from the IgG class. If the chemical group A is a hapten, for example, then the affinity substance is an antibody which was obtained against the hapten, for example by immunization with hapten conjugates. The remaining Combinations and variations of the respective assignments are readily apparent to the person skilled in the art. It goes without saying that the specific list is not exhaustive.

For the detection of the substance to be detected by means of the immunoassay according to the invention there are marked affinity substances which, if they do not have their own measurable characteristics, in turn have groups which, for example, contain radioactive isotopes or chromophores and / or fluorophores or bound enzymes that can cause color reactions.

The principle of the invention is explained in more detail below using a specific exemplary embodiment. The detector molecule (a polypeptide) carries biotin as chemical group A.

The high affinity binding of the antigen with a predetermined binding site to the microtiter plates results in a defined alignment of the antigen and ensures the defined accessibility of all epitopes of the antigen. Such binding is achieved via a streptavidin-biotin system. For this purpose, antigens are selectively synthesized with a biotin molecule at the N-terminal or at the C-terminal end, or at another point on the molecule. The microtiter plates coated with streptavidin ensure high affinity binding (affinity constant IO ^"15 mol / 1) of the biotin-peptide complex (Chaiet, L., Wolf, FJ (1964), Arch. Biochem. Biophys. 106, 1-5). This results in a strong and complete binding of the antigen to the microtiter wall (figure). Desorption during the execution of the assay, as occurs in the classic assay system without biotin due to unspecific hydrophobic interactions, does not occur therefore lower than a direct immobilization. Binding comparisons between the classic immobilization assay, in which the antigen with an unpredictable binding site but unspecific hydrophobic interactions was bound to the microtiter wall, and the streptavidin-biotin immobilization assay, in which the N- or the C-terminal end of the antigen biotinylated and the biotin epitope was subsequently bound to the microtiter wall with high affinity via streptavidin, resulting in a significantly higher sensitivity of the streptavidin-biotin assay compared to the classic assay. The displacement by adding unbound antigen was surprisingly carried out in the streptavidin-biotin assay as an embodiment of the immunoassay according to the invention even at significantly lower concentrations than in the classic immobilization assay.

The present invention also relates to a detector substance which can be used, inter alia, in the immunoassay according to the invention, but opens up further possible uses.

The affinity substance according to the invention contains a mixture of at least two different detector molecules, the at least two detector molecules each having a chemical group A at different positions, which is suitable for entering into an affinity bond, the chemical group A at a different but specific position in each case Detector molecules is bound.

Preference is given to using a detector substance which contains a polypeptide which is in each case specifically labeled at different positions. In principle, the chemical groups A mentioned above come into question. The use of biotin which is bound to amino groups (for example by lysine) occurring in the amino acid sequence of the polypeptide is particularly preferred. According to the invention, the decisive factor here is the respective biotinylation for the at least two different detector molecules but to be introduced specifically so that two groups of detector molecules are present. One group has, for example, the biotinylation at the N-terminus, whereas the second group of detector molecules has the biotin label specifically at another location, preferably the C-terminus. It can be seen that this description is only exemplary. In principle, every possible position comes into question. However, if a position has been selected, this position should be maintained for the class of the detector molecules, so that the advantages according to the invention, which are achieved by using the at least two different detector molecules, are fully realized. The detector substances according to the invention can be used in different areas. For example, the detector substances in immunoassays can also be used for the detection and quantification of antibodies, autoantibodies and endogenous substances, in particular regulatory peptides. They can also be used for receptor labeling as a diagnostic in fluorescence microscopy and flow cytometric diagnosis of diseases which are associated with the expression or change in the expression of receptors. such as carcinoid diagnostics at the level of somatostatin receptors.

Furthermore, the detector substances according to the invention can be used in the characterization of unknown receptors of known peptides and in the search for receptor analogs by means of fluorescence microscopy and flow cytometry. The detector substances are also suitable for the purification of peptides by means of affinity chromatography. Antibodies can be purified, for example, if the detector substances according to the invention are bound to a solid phase matrix via their affinity group A. It is also possible to use the detector substances according to the invention in medical diagnostic tests, in particular using biosensors.

Another area of application of the detector substance according to the invention is flow-cytometric analytical and preparative tive separation of complex cell mixtures, the cells being characterized by surface or intracellular labeling.

Obtaining an effective antigen-specific antibody is important for the successful development of an immunoassay kit. Antibodies can be specifically characterized with the help of immobilized biotinylated peptides, the binding site and the epitope presentation of which can be predetermined based on the biotinylation.

By binding biotinylated peptides to microtiter plates, the titers of the corresponding antibodies or autoimmune antibodies in the blood can be determined according to the principle of the immunization immunoassay.

In the search for the binding sites of active substances in the organism, it was previously necessary to add detectable markers to the active substances, e.g. with radioactivity or with fluorescence. However, these markers can hide the epitopes of the active substance for the receptor binding sites and thereby reduce the affinity of the receptor for the active substance. The incorporation of biotin into the drug molecule, on the other hand, can be controlled very precisely, as a result of which the epitopes of the drug remain accessible to the receptor binding sites. The drug receptors in the organism initially bind the free epitopes of the biotinylated drug. To visualize the binding, the in-vitro or in-vivo system is e.g. Fluorescence-labeled streptavidin added. The high affinity of streptavidin for biotin leads to the binding of the fluorescence-labeled streptavidin to the biotinylated active substance bound by the receptor. In this way, the complex of receptor-active substance-biotin-streptavidin fluorescence can be localized for the observer.

In the fluorescence microscopy and flow cytometric diagnosis of diseases that are associated with the expression or the change in the expression of receptors biotinylated peptides are used for receptor labeling, for example in carcinoid diagnostics at the level of somatostatine receptors.

Antibodies generated by immunization are by no means uniform, but always consist of a mixture of different antibody species, which also differ in their regioselectivity towards the different epitopes or molecular areas of the antigen. The high affinity binding of biotin to streptavidin can be used for the purification and differentiation of antibodies - based on the selective incorporation of the biotin by chemical methods at the C-terminus, at the N-terminus, or also in the central position of the antigen. Antigens selectively labeled with biotin - or derivatives derived from the structure of the antigen - can be attached to a matrix, e.g. a hydrophilic chromatography material, to which streptavidin is bound, can be immobilized with a defined spatial orientation. This method ensures that affinity chromatographic purifications are carried out with improved monitoring of the interaction between antibody and antigen. In addition, the defined loading of the chromatography column with a biotinylated antigen permits a higher loading of the affinity matrix. In addition to increasing the selectivity of an antigen-antibody interaction, this can also lead to a quantitative increase in the regioselective antibodies obtained.

The invention is explained in more detail by the following specific embodiment.

A basis of the method according to the invention is the synthesis of peptidyl resins and peptides blocked with protective groups, which can optionally be selectively linked to biotin or its derivatives at certain amino acid positions of the peptide chain. It is possible that both after peptide synthesis using Fmoc chemistry and Boc chemistry in the totally blocked peptidyl resin, the N-terminal amino group below Preservation of the remaining protective groups can be selectively split off. This regenerated amino group can then be converted by known coupling methods in peptide chemistry, but preferably with uronium or phosphonium compounds of the TBTU or BOP type, with biotin or its derivatives to give the N-terminal biotin-labeled peptidyl resin. The corresponding N-biotinyl peptides can be obtained in high purity by subsequent cleavage from the support and the other protective groups and subsequent preparative chromatographic purification.

Surprisingly, selective biotinylation at the C-terminus of peptides is preferably achieved by using the lysine derivative N- (9-fluorenylmethoxycarbonyl) -N- [1- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl] -L-lysine [Fmoc-Lys (Dde) - OH]. If the peptides in question do not contain lysine as the C-terminal amino acid in their sequence, the solid phase synthesis is carried out starting with Fmoc-Lys (Dde) -OH as the C-terminal building block. After the peptide synthesis has been carried out, the Dde group can be split off in the fully protected peptidyl resin or peptide by repeated treatment with dilute hydrazine hydrate, all other protective groups of the peptide being retained. After coupling with biotin or its derivatives by known activation methods of peptide chemistry and subsequent unblocking and cleavage from the carrier, selectively biotinylated peptides can be obtained in highly pure form at the C-terminus.

An advantage of this process is the fact that the introduction of the biotinyl residues can be carried out in high yield after the entire peptide chain has been synthesized. This applies in particular to the display of C-terminal biotin-labeled peptides. Surprisingly, the biotin residue can be introduced in high yield at the sterically unfavorable resin-bound amino acid position.

In contrast to methods according to the prior art of peptide synthesis, for linking biotin or its derivatives with the selectively unblocked amino group at the N- or C-terminus Only the solvent 1,3-dimethyl-3,4,5,6-tetrahydro-2 (IH) -py-rimidinone (DMPU) is particularly preferred. This allows simple dissolution of biotin or its derivatives in high concentration and their linkage with the peptide terminal amino group in high yields, even in excesses of three equivalents.

The (+) biotin can be selectively introduced into the protected peptidyl resin in the form of the natural compound or its carboxyl-activated active esters and surprisingly also as a 6-aminocaproic acid or lysine-spaced derivative using the methods described.

The synthetic biotinyl-labeled peptides can be identified by various analytical methods. These include methods of chromatography, capillary electrophoresis, mass spectrometry, peptide sequencing and amino acid analysis.

With regard to the present invention, it is worth mentioning, for example, the display of urodilatin which is selectively labeled at the N- and C-terminus as well as correspondingly labeled fragments of the human parathyroid hormone. Urodiline has special clinical applications. In an ELISA test (enzyme-linked immunosorbent assay), its biotinylated derivatives are of great importance for the analysis of the formation of human autoantibodies against the peptide.

The assay according to the invention is preferably implemented by binding the C- and N-terminal biotin-labeled synthetic peptides to microtiter plates loaded with streptavidin. After this binding, it is possible to bind peptide-specific polyclonal or monoclonal antibodies to the immobilized peptide. It is thus advantageously possible to measure both antibodies which are directed against C-terminal regions of the respective peptide and antibodies which are directed against N-terminal regions of the peptide in a single assay, by using both synthetic biotinyl peptides can be used in a test. The corresponding test can also be carried out separately. This makes it possible to detect antibodies against the C- and N-terminus of peptides in separate analyzes.

In both cases, the bound peptide-specific antibodies are characterized by protein A, protein G or a secondary antibody, e.g. B. an anti-human IgG antibody, is bound to the species-specific Fc fragment of the human antibody.

According to the invention, protein A or protein G can be labeled, for example radioactive, to quantify the peptide-antibody binding. The secondary antibody can be conjugated to an enzyme or to radioactively labeled substances. The enzymes are able to convert certain substrates in a photometrically traceable reaction. According to the invention, peroxidases or phosphatases, for example, can be bound to the secondary antibody. For example, tetramethylbenzidine and 2,2 '-azino-bis- (3-ethylbenzthiazoline-6-sulfonic acid (ABTS) for peroxidases or 4-methylumbilliferyl phosphate, p-nitrophenyl phosphate and bromochloroindolyl phosphate-nitro-blue-tetrazolium for The reaction products of these substrates can be measured by various methods, but above all by measuring the fluorescence or the color intensity of the reaction products. The radioactivity conjugated to protein A, protein G or the secondary antibody is determined in the gamma counter. The fluorescence or The photometrically measurable fluorescence or staining or the radioactivity corresponds quantitatively via the secondary antibody to the amount of the peptide-specific antibody which is bound to the specifically N- or C-terminally biotinylated peptide of on the selective synthesis of Measurement methods based on N- or C-terminal biotinylated peptides are shown in FIG. According to the invention, peptides (eg urodilatin) which are immobilized in this way do not lose their antigenic properties. The work also shows that the antigenic properties of the peptides biotinylated selectively at the N- or C-terminus are still present even after drying and reactivation with various assay buffers. The antigenic activity of the N-biotinyl peptides does not decrease by storing the dried microtiter plate over a period of several weeks.

The synthesis of selectively labeled biotinyl peptides takes place, for example, by producing partially unblocked peptidyl resins according to the Merrifield solid phase method, both syntheses using the fluorenylmethoxycarbonyl (Fmoc) and the tert-butyloxycarbonyl (Boc) group as temporary amino protecting group are suitable (E. Atherton and RC Sheppard, Solid Phase Synthesis, IRL Press, Oxford 1989; J. Jones, The Chemical Synthesis of Peptides, Clarendon Press, Oxford 1991).

Example 1: Preparation of peptides with selective N- or

C-terminal (+) biotinyl label

To produce N-terminally biotinylated peptides, the peptide syntheses are carried out according to methods known per se. Fully protected peptidyl resins prepared with Fmoc chemistry are then treated for 10 minutes with a solution of 20% piperidine in N, N-dimethylformamide (DMF) and then washed carefully with DMF. Peptides synthesized according to Boc chemistry are treated after the last amino acid coupling for 30 minutes with a solution of 50% trifluoroacetic acid in dichloromethane and washed carefully with dichloromethane. Alternatively, in the case of peptide synthesis by Boc chemistry, the last amino acid can also be introduced as an Fmoc derivative. Its amino group can also be selectively released by 20% piperidine in DMF. The peptidyl resins obtainable in this way have a free amino function at the N-terminus and can thus be selectively linked to biotin or its suitable derivatives to form the biotinyl-labeled peptide. For this linkage, biotin itself and its carboxyl-activated derivatives, such as, for example, the N-hydroxysuccinimide ester, the 4-nitrophenyl ester and the hydrazide, and preferably N-biotinyl-6-aminocaproic acid and its carboxyl-activated derivatives, such as the hydrazide, can be used and the N-hydroxysuccinimide ester can be used. Before being linked to the N-terminal amino group of the protected peptidyl resin, the biotin or its derivative is dissolved, preferably in N, N-dimethylformamide, N-methylpyrrolidone, trifluoroethanol, dimethyl sulfoxide or 1,3-dimethyl-3,4, 5, 6-tetrahydro-2 (1H) pyrimidinone (DMPU), and treated with 1.1 equivalents of activator. Suitable activators are 2 (IH) -benzotriazol-l-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate (TBTU) and compounds derived therefrom, benzotriazol-1-yl-oxy-tris- (dimethyamino) -phosphonium hexafluorophosphate (BOP) and compounds derived therefrom as well as N, N-dialkylcarbodiimides such as diisopropylcarbodiimide (DIC) and dicyclohexylcarbodiimide (DCC) with the addition of other auxiliaries such as N-hydroxysuccinimide, 1-hydroxybenzotriazole or diisopropylethylamine. To biotinylate the amino group, the biotin or its derivative is preactivated for 10 minutes at room temperature and then combined in a two to ten-fold excess based on the peptidyl resin with the latter for 10 to 20 hours with shaking. The biotinylated peptidyl resin is then carefully washed with the solvent, 2-propanol and dichloromethane used to remove excess reagents and dried under high vacuum. The peptide labeled in this way is cleaved from the carrier resin within 90 minutes by adding preferably trifluoroacetic acid / ethanedithiol / water (94: 2: 2, volume), freed from the protective groups, precipitated with cold tert-butyl methyl ether and lyophilized . The isolated crude product is purified by preparative high-performance liquid chromatography and then characterized. Compared to the non-biotinylated peptide, the biotin-labeled peptide always has a significantly higher retentivity when C18 reversed-phase chromatography is carried out. tion time. The N-terminally bound biotin can be detected by mass spectrometry by means of a molecular weight which is 226.3 daltons larger. When using N-biotinyl-6-aminocaproic acid, the molecular weight increases by 339.5 daltons.

Selective biotin labeling can be achieved with special lysine derivatives both in the context of the Fmoc and the Boc synthesis strategy. If no lysine is present at the C-terminus of the peptide sequences to be biotinylated, the peptide synthesis is started with an additional lysine, which is followed by the actual peptide chain of the desired substrate. Within the framework of Fmoc chemistry, the protected lysine derivative N- (9-fluorenylmethoxycarbonyl) -Nl- (4,4-dimethyl-2-6, dioxocyclohex-l-ylidene) - ethyl-L-lysine [Fmoc-Lys ( Dde) -OH] (BW Bycroft, WC Chan, SRChadra, ND Hone (1993) J. Chem. Soc, Chem. Commun. 1993, 778; HF Brugghe, HAM Timmermans, LMA van Unen, GJ ten Hove, G van de Werken, JT Poolman, P. Hoogerhout (1994) Int. J. Pept. Protein Res. 43, 166). After the peptide synthesis has ended, its Dde protective group is split off, as a result of which the amino group of the C-terminal lysine is released. For this purpose, the completely blocked peptide, which preferably carries a Boc group as the N-terminal amino protective group, is reacted three times for two to 15 minutes with a solution of 2 to 10% hydrazine hydrate in N, N, dimethylformamide with shaking. When carrying out a peptide synthesis according to the Boc strategy, the derivative N- (tert-butyloxycarbonyl) -N- (9-fluorenyloxycarbony) -lysine [Boc-Lys (Fmoc) -OH] is used. After the peptide synthesis is complete, the amino group of the C-terminal lysine can be released for 5 to 15 minutes by adding 20% piperidine in N, N-dimethylforinamide. After selective deblocking of the C-terminal lysine, both resins are carefully washed with N, N-dimethylformamide.

Carrying out the selective C-terminal coupling with biotin or its derivatives, splitting off the carrier resin and deblocking, preparative purification of the biotinyl peptide and its characterization is carried out using the same methods as described for the N-terminal biotinylated peptides.

The method can in principle be used for all carrier-bound synthetic peptides which are selectively labeled with biotin or its derivatives at the N- or C-terminus by the method of the protective group technique according to the invention. The method particularly includes the chemical synthesis of selectively biotinylated peptides derived from urodilatin (CDD / ANP-95-126) and other natriuretic peptides as well as from human parathyroid hormone and its fragments.

By combining the two protecting group techniques for the selective labeling of the N- and the C-terminus of any peptides, the method also includes peptidyl resins and peptides which are selectively labeled at the N- and the C-terminus by biotin.

Example 2: Uses of synthetic, selective (+} -

Biotin labeled peptides

The synthetic, selectively (+) -biotin labeled peptides are immobilized by coupling to a streptavidin-coated microtiter plate via a biotin-streptavidin bond.

When performing the assay, the microtiter plate is washed with a wash solution (e.g. phosphate buffer; phosphate buffer: 136 mM sodium chloride; 2.68 mM potassium chloride; 10 mM sodium dihydrogen phosphate; 1.76 mM potassium dihydrogen phosphate) between each work step.

The microtiter plate is incubated with a streptavidin solution (eg 20 μg / ml) for two hours. The free binding sites are then saturated with a blocking buffer (eg 3% bovine albumin in phosphate buffer or 2.5% casein, 0.2% Tween 20 in phosphate buffer) for at least 2 hours. The biotinylated peptides (e.g. N- and C-terminal biotinylated Urodilatin) are dissolved in 1 M acetic acid (eg 30 μg / ml) and introduced into the wells of the microtiter plate coated with streptavidin. After an incubation period of one hour, the excess biotinylated peptides are washed out. The serum to be examined is either neat or diluted with a physiological buffer (eg 3% bovine albumin in phosphate buffer; 1: 250; urodilatin antibody serum: physiological buffer) pipetted onto the microtiter plate and incubated for 2 hours. The secondary reagent (for example conjugated or iodinated anti-human IgG antibody or iodinated protein A or iodinated protein G or conjugated with peroxidase or alkaline phosphatase) is then added to the microtiter plate and incubated for 2 hours at room temperature. After the excess secondary reagent has been washed out, the radioactivity can be measured directly in the gamma counter. When using enzyme-labeled secondary reagents, a solution of a chromogenic substance (for example 2,2'-azino-bis- (3-ethylbenzthiazolin-6-sulphonic acid (ABTS), 4-methylumbilliferyl phosphate) is pipetted in. This is added to an enzyme-dependent reaction to form a quantitatively measurable, fluorescent or colored substance. The fluorescence or the color intensity is determined in the spectrophotometer.

The amount of radioactivity or the resulting fluorescent or colored substance is directly proportional to the amount of the antibody that has bound to the biotinylated peptide.

Claims

Patent claims

1. Immunoassay with a detector substance that interacts with a substrate to be detected, where

the detector substance is a mixture of at least two different detector molecules, each of which carries a chemical group A at different positions, which is suitable for forming an affinity bond,

the detector substance consisting of at least two detector molecules is brought into contact with a sample which contains the substrate to be detected, after the formation of complexes from the substrate to be detected and the two different detector molecules, the complexes being formed by an affinity substance which is with the chemical group A forms an affinity bond, is immobilized and/or detected.

2. Immunoassay according to claim 1, characterized in that the respective detector molecule is a polypeptide, such as antibodies, a glycosylated peptide, such as lectins, an oligosaccharide or polysaccharide, nucleic acids and / or haptens.

3. Immunoassay according to claim 1 and / or 2, characterized in that the chemical group A, which is suitable for forming an affinity bond, is biotin, streptavidin, an antigen, an antibody, protein A, protein G, F _c fragments, F _ab fragments, hormones, hormone receptors, enzymes, enzyme substrates and/or nucleic acids.

1. Immunoassay according to claim 1 and / or 2, characterized in that the affinity substance is complementary to the chemical groups A mentioned in claim 3.

5. Immunoassay according to at least one of claims 1 to 5, characterized in that the affinity substance is labeled, for example with radioactive isotopes or with chromophores and/or fluorophores.

>. Detector substance containing a mixture of at least two different detector molecules, which at least two detector molecules carry a chemical group A at different positions, which is suitable for entering into an affinity bond and where the position of the chemical group A is specific for each of the different detector molecules.

Detector substance according to claim 6, characterized in that the detector substance consists of at least two polypeptides whose amino acid sequence is the same and carry a chemical group A at different positions.

Detector substance according to claim 7, characterized in that the chemical group A is biotin and is specifically located at the N-terminal end of one detector molecule and at the C-terminal end of the other detector molecule, so that the two different detector molecules on the one hand N-terminal carry the chemical group and the other group of the detector molecule carry the chemical group A at the C-terminal end.

9. Use of a detector substance according to at least one of claims 6 to 8 in an immunoassay

for the detection and quantification of antibodies, autoantibodies and endogenous substances, in particular regulatory peptides, for receptor labeling as a diagnostic in fluorescence microscopy and flow cytometric diagnosis of diseases that are associated with the expression or the change in the expression of receptors, e.g. Carcinoid diagnostics at the level of somatostatin receptors, for the characterization of unknown receptors of known peptides and in the research of receptor analogues using fluorescence microscopy and flow cytometry.

10. Use according to claim 9 for the purification of antibodies by affinity chromatography, wherein the detector substance is bound to a solid phase matrix via the chemical group A.

11. Use according to claim 9 for the flow cytometric analytical and preparative separation of complex cell mixtures by surface or intracellular labeling of proteins.

12. Diagnostic agent containing a detector substance according to one of claims 6 to 8.