KR19980064214A - Immunodissociation for Improving Immunochemical Measurements of Analytes - Google Patents

Abstract

The present invention relates to an immobilized specific receptor R1 which exhibits an interactive biocompatibility with the analyte, and likewise one or more of the samples using a specific receptor R2 that exhibits interactive biocompatibility with the analyte. A method for immunochemically measuring analytes. In the novel method, receptor R3 having at least one specific binding site is added to the analyte, and the immune complexes thus obtained are completely or partially dissociated, after which they recombine and continue to be detected. According to a further preferred embodiment, in addition to receptor R3, receptor R4 having affinity for R3 and immobilized on a solid phase is used to dissociate the immune complexes thus obtained completely or partially, after which they recombine and continue to be detected.

Description

Immunodissociation for Improving Immunochemical Measurements of Analytes

The present invention relates to an immobilized specific receptor R1 which exhibits an interactive biocompatibility with the analyte, and likewise one or more of the samples using a specific receptor R2 that exhibits interactive biocompatibility with the analyte. A method for immunochemically measuring analytes. In the novel method, receptor R3 having at least one specific binding site is added to the analyte, and the immune complexes thus obtained are completely or partially dissociated, after which they recombine and continue to be detected. According to a further preferred embodiment, in addition to receptor R3, receptor R4 having affinity for R3 and immobilized on a solid phase is used to dissociate the immune complexes thus obtained completely or partially, after which they recombine and continue to be detected.

Conventional immunological methods for diagnosing a disease inducer, eg, a virus, bacterium, allergen, autoantigen or disease associated with the formation of specific antibodies to a drug in particular, have an antigenic structure in which these antibodies belong to the inducer. Based on the ability to form complexes.

In particular in the embodiment of the method, generally referred to as heterologous immunoassay, the sample to be investigated for its component, for example a specific antibody (analyte antibody), is immobilized on the antigenic structure of the disease causing agent, on a suitable supporting material known in the art. The antigenic structure thereof. The analyte antibody present in the sample is detected by binding to the antigenic structure of the disease causing agent immobilized to the support material as an immune complex. Detection antibodies or other specific receptors (eg, Protein A) that can form a complex with the analyte antibody of the sample can be used for detection.

Typically, the detection reagent carries a label that enables the detection of the amount of antibody bound methodologically.

Commonly used labels are radioisotopes, enzymes, fluorescent materials, phosphorescent or luminescent materials, materials with stable unpaired electrons, latex particles, magnetic particles, metal colloidal solutions and red blood cells.

It is known that the method comprises both single-step and multi-step detection methods. Each procedural step usually ends in a separation process (washing step).

However, the technique of the single-step method, which is performed very simply in heterogeneous immune assays, is not suitable for detecting all disease markers. Two- or multi-step methods have often been applied for technical reasons.

However, multistage methods called immune complex transfer enzyme immune assays are also known. S. Hashida et al., Journal of Clinical Laboratory Anaysis 8: 86-95 (1994). In this method, the complete immune complex comprising the solid phase antigen, the specific antibody and the labeled combination antigen is separated from the solid phase. Complete immune antibodies are then pipetted onto antibody-binding solids to immobilize and detect.

The method is very specific, but if the disease causing agent or antibody directed against it is introduced into the complex with the specific receptor immobilized in the first step, subsequent reaction steps are carried out in reverse reaction known to those skilled in the art. Can be partially separated from the complex again, resulting in the exclusion of the detection reaction, resulting in reduced reactivity.

The diagnostic efficiency of the multistep method is particularly reduced when the rate of reverse reaction is high between the immobilized receptor and the substance to be detected. Such high rates are obtained, for example, for antibodies with low affinity for disease causing agents or agents. The skilled person knows that the effect is particularly obtained in the case of methods of frequently detecting variant disease causing agents or disease markers which are subsequently mutated to show only a weak interaction with the immobilized specific receptor.

EP 0 572 845 states that the rate of reverse reaction is substantially reduced by adding additional receptors on the structural features of the material to be detected. This receptor must have one or more binding sites for the substance to be detected and must not interfere with the immunochemical detection of the substance.

However, despite the marked reduction in adverse reactions, the method cannot likewise be used to reliably and with high reactivity detect particular low-affinity antibodies to disease agents or agents.

Therefore, it is an object of the present invention to identify reagents that do not have this disadvantage.

Surprisingly, an immune complex already formed, consisting of immobilized specific receptor R1, analyte A which interacts with receptor R1, and a specific receptor R2 directed and commonly labeled for the analyte, dissociates immune By performing the step and subsequent recombination steps, it was found that an increase in signal could be seen when additionally receptor R3 was added. The latter receptor does not inhibit the immune response between Analyte A and Receptor R1 or Analyte A and Receptor R2 and reduces the proportion of analytes that do not bind to the solid phase because they can bind to one or more analytes.

In another embodiment additional immobilized receptor R4 is added in addition to R3, and receptor R4 may exhibit interactive biocompatibility with R3. R4 reduces the proportion of unimmobilized immune complexes by binding an immune complex consisting of analyte A and receptor R3 to the solid phase. In this case, however, the specific receptor R3 does not have to have one or more specific binding sites for analyte A as in the above embodiments. This additional binding of analyte A and generally the receptor R2 with labeling results in a marked increase in signal yield and ultimately an increase in reactivity of the assay for analyte A.

In another embodiment, the receptor R4 is immobilized on the secondary solid phase, and the dissociated immune complex, such as R3-A-R2, transitions from the primary solid phase to the secondary solid phase, where a recombination step and detection of R2 also occur.

Analyte A detected within the meaning of the present invention may be, for example, an antigen such as an antibody induced by a disease causing agent or the disease causing substance itself.

Therefore, the present invention relates to a specific receptor R1 immobilized on a support and to which the extent of binding of the analyte to the specific receptor R1 is measured directly or indirectly with additional specific receptor R2 carrying a label, and the substance A to be detected. Degree of binding of analyte A to specific receptor R2 having one or more binding sites, having no specific affinity for immobilized specific receptor R1, adding unlabeled receptor R3, and directly or indirectly carrying a label It relates to a method of immunochemically measuring one or more analytes using the method of measuring the following recombination.

The invention also relates to a specific receptor R1 immobilized on a support and to which the extent of binding of the analyte to the specific receptor R1 is determined directly or indirectly with a further specific receptor R2 carrying a label, and the substance to be detected in the dissociation step. In addition to binding factor R3, which should not have more than one binding site for the antibody, additional specific immobilization or immobilized receptor R4 is added, which receptor R4 shows affinity for R3 and is directly or indirectly accompanied by a label. A method for immunochemically measuring one or more analytes using a method of measuring the extent of binding of analyte A to receptor R2 by the following recombination.

The invention further comprises a specific receptor R1 immobilized on a support, wherein the degree of binding of the analyte to the specific receptor R1 is determined directly or indirectly by a further specific receptor R2 carrying a label, and a dissociation step. In other than binding factor R3, which should not have one or more binding sites for the substance to be detected, the dissociated translation complex, eg, R3-A-R2, further comprises a specific immobilizable or immobilizable receptor R4. Transferring to the secondary solid phase, the receptor R4 shows affinity for R3, using a method to determine the extent of binding of analyte A to specific receptor R2 directly or indirectly with labeling by the following recombination A method for immunochemically measuring one or more analytes.

In the sense of the present invention, unlabeled means that R3 does not involve a specific label or a label used to determine the extent of analyte binding to a specific receptor.

In the method the specific receptor R4 can be known and used to the skilled person in all its embodiments. An important factor is preferably any immunity to which the analyte binds to specific receptors immobilized immunochemically or similarly in the first step and is detected either directly or indirectly in the second step but not necessarily in separate steps. In the chemical method, a novel method can be applied in a suitable form.

The method may be applied to a luminescent label, preferably used by a skilled person in the art known as a sandwich ELISA, preferably as an enzyme or labeling system with a dyeable or fluorescent substrate. However, the selected embodiment does not have a significant impact on the possible use of the new method.

A test modulus comprising a test element having a fine titration plate, magnetic particles, latex particles or a chemical matrix, for example a fiber or a membrane, is preferably used in the solid phase.

For those skilled in the art, as mentioned above, immunological methods can be performed by different analytes, such as HIV 1 (HIV = human immunodeficiency virus) and HIV 2, respectively or in combination with each other, or by combining HIV 1 and / or HIV 2 with HCV ( hepatitis C virus or hepatitis C virus antigens) and are known to be applicable for simultaneous measurement. In the meaning mentioned above, the analyte may be a viral antigen or an antibody directed against it. Such embodiments are also included herein.

Dissociation in the sense of the present invention includes all chemical and physical methods that can disrupt or reduce biocompatible interactions in reverse. The skilled person is known as chemicals used in immunochemical purification of proteins and antibodies, for example thiocyanate, urea or guanidine. Dissociation can also occur by immunological methods such as competition with immunologically suitable agonists. Dissociation can further be carried out using an acidic or alkaline solution. Similar to the skilled person, the physical method of dissociation is known, for example, by temperature difference, electromagnetic waves or ultrasonic waves.

Recombination within the meaning of the present invention is to non-dissociative conditions, such as neutralization or dilution, or simply to the inverse of previously performed dissociation conditions (e.g. to remove the above-mentioned temperature differences or to stop the electromagnetic wave generator or ultrasonic generator). Means to do.

Receptor R3 within the meaning of the present invention is a specific binding partner having at least one biocompatible binding site for analyte A. In addition, the receptor R3 can be introduced by interaction of the receptor R4. The binding partner or component of the receptor R3 may be a conjugate of the antibody or the antibody itself. The antibody or antibody fragment or conjugate thereof may be direct to A or other A and R4. Receptor R3 may also include an antibody or antibody fragment to which receptor R4 is coupled to the structural element indicated.

Receptor R4 within the meaning of the present invention is a specific binding partner having one or more binding sites for R3. The receptor or component of the receptor may be a conjugate of the antibody / antibody fragment or the antibody / antibody fragment itself. The antibody / antibody fragment or conjugate thereof may be direct to R3 or may have an antigenic structure that is recognized as a binding site comprising receptor R3 in addition. R 4 also includes a protein to which R 3 is coupled to the structural element indicated.

The invention also relates to a reagent for use in the above-mentioned method.

Preferred combinations of receptors for detecting specific antigens (analytes) are

R1: Antibody directed against analyte

R2: antibody with label and directed against the analyte

R3: is the antibody directed against Analyte A.

Preferred combinations of receptors for detecting specific antigens (analytes) are

R1: Antibody directed against Analyte A

R2: antibody with label and directed against analyte A

R3: Antibody directed against Analyte A

R4: is an antibody directed against antibody R3.

If the antigen is detected, the antibody or antibody fragment does not recognize the same epitope in which the solid phase R1 or binding antibody R2 is preferably used as the receptor R3.

Preferred combinations of receptors for detecting specific antibodies (analytes)

R1: Antigen directed to Analyte A

R2: antigen with label and directed to Analyte A

R3: Antibody directed against Analyte A

Preferred combinations of receptors for detecting specific antibodies (analytes)

R1: Antigen directed to Analyte A

R2: antigen with label and directed to Analyte A

R3: Antibody directed against Analyte A

R4: is an antibody directed against antibody 3.

Particularly preferred combinations of receptors for detecting specific antibodies (analytes) are

R1: Antigen directed to Analyte A

R2: antigen with label and directed to Analyte A

R3: Antibody directed against Analyte A and carrying an additional epitope that is not antibody-specific

R4: Indicated for a further epitope of antibody R3, wherein the epitope is an antibody that is not antibody-essential.

The skilled person is known as a method for preparing a conjugate comprising in each case an antibody which is not antibody-essential and additional antigens (eg biotin or digoxygenin). While retaining the biocompatible functions of the starting materials, the conjugates can be prepared by linker or biocompatible interactions, for example with the aid of chemical reagents. Hybrid molecules can also be prepared by chemical synthesis, hybridoma technology, or recombinant DNA methods.

The novel reagents can be used in many human and veterinary diagnostics. Examples cited include detecting antibodies of different immunoglobulin species against viruses (eg hepatitis A, B and C viruses and also different HIV types), bacterial and parasitic etiology and also structural features of allergic diseases. It's a test. Other crises are bill disease-inducing substances such as viruses (eg hepatitis B virus), bacteria, parasites and allergens, and also disease markers (eg tumor markers) in multi-step detection methods.

The invention is apparent in the following examples and claims, but is not intended to further limit the particulars of the more general teachings.

Example 1

1a) Preparation of solid phase

Type B microtiter plates (purchased from Nunc, Roskilde, Denmark) were prepared using a coating solution (50 mM sodium carbonate buffer, recombinant gp41 [Behringwerke AG, Marburg, FRG] 600 μg / L and biotin [Behringwerke AG, Marburg, FRG] in pH 9.5. Incubate for 24 hours at 4 ° C. with 100 μl per well of monoclonal antibody). The wells of the microtiter plates are then washed three times with 300 μl of wash solution (50 mM Tris, 0.1% Tween 20, pH 7.2). Microtiter plates dried with silica gel are stable for about 1 year under anaerobic conditions.

1b) Preparation of the Binder

10 mg of HIV 1 gp41 peptide (IAF Biochem, Laval, Canada) is dissolved in 1 ml of anhydrous acetic acid / water (50:50, v / v). After neutralization with 5N sodium hydroxide solution, 10 times more molar N-γ-maleimidobutylylsuccinic acid is added and the mixture is stabilized for 1 hour at room temperature. Non-reacting heterobifunctional reagents are separated by gel filtration (Sephadex G-25) using 100 mM sodium phosphate, nitrilodriacetic acid 5M, pH 6.0.

10 mg of horse radish peroxide (Boeringer Mannheim, Mannheim, FRG) is incubated with 100 times greater molar 2-iminothiolane in 10 ml of 10 mM sodium phosphate, 100 mM sodium chloride pH 8.0 for 1 hour at room temperature. The free modifying reagent is then removed by gel filtration (Sephadex G-25) using 100 mM chloride phosphoric acid, 5 mM nitrilotriacetic acid, pH 6.0.

Two eluents (SH-activated peroxidase and maleimide-modified HIV 1 peptide) are combined and incubated overnight at room temperature. After the reaction was stopped with 1/10 vol of 100 mM N-ethylmaleimide, the conjugate was removed by gel filtration (Sephadex G-25) from the unreacted HIV 1 peptide. After concentration (2 mg / ml), the binder is stored at -20 ° C.

1c) Preparation of Biotinylated Antibodies

10 mg of monoclonal antibody against human immunoglobulin G (Behringwerke AG, Marburg, FRG) dissolved in 10 mM sodium phosphate, 100 mM sodium chloride, pH 8.0 was dissolved in 10 mM sodium phosphate, 100 mM sodium chloride, pH 8.0, 10 ml. Incubate with N-hydroxysuccinimide-X-biotin for> 10 fold molar for 1 hour at room temperature. The free modifying reagent is then removed by gel filtration (Sephadex G-25) using 100 mM Tris, 5 mM nitrilotriacetic acid, pH 7.0.

Example 2 Use of New Reagent

2a) Enzyme Immunoassay to Detect HIV 1 Antibody (Reference System I)

Enzyme immunoassays for detecting anti-HIV 1 antibodies are performed as follows.

25 μl of sample buffer (0.3 m Tris / HCl, 1% albumin, 2% Tween 20, pH 7.2) was added at 37 ° C. with 100 μl of human serum in a well of a microtiter plate prepared as described in Example 1a above. Incubate for 30 minutes at. Plates were washed four times with 50 mM PBS, 0.1% Tween 20 and conjugates prepared as described in Example 1b (0.1M Tris / HCl, 1 mM glycine, 0.2% albumin, 0.4% fluronin F64, human immunoglobulin G 125 μl of a monoclonal antibody against 1: 1000 at pH 8.1) is pipetted into each well. The 30-minute incubation (+ 37 ° C.) is terminated with four additional wash steps. Binding peroxidase activity that directly correlates with a number of binding anti-HIV antibodies is measured by adding H ₂ O ₂ / tetramethylbenzidine. After 30 minutes at room temperature, substrate conversion is stopped by addition of 0.5 M sulfuric acid. Absorption is measured at 450 nm.

2b) Enzyme Immunoassay to Detect HIV 1 Antibody (New System I)

Enzyme immunoassays to detect HIV 1 antibodies are performed as follows.

25 μl of sample buffer (0.3 M Tris / HCl, 1% albumin, 2% Tween 20, pH 7.2) was added at 37 ° C. with 100 μl of human serum in the well of a microtiter plate prepared as described in Example 1a above. Incubate for 30 minutes at. The plate is washed four times with 50 mM PBS, 0.1% Tween 20 and 100 μl of dissociation buffer (5 mM glycine / HCl, pH 2.5) is added at RT for 30 minutes. Binder (0.5M Tris / HCl, 1 mM Glycine, 1% Albumin, 2% Pluronin F64, Monoclonal Antibody Against Human Immunoglobulin G, Prepared as described in Example 1b, 1 at pH 8.1) : 200 dilution) 25 μl are pipetted into each well without washing step. The 30-minute incubation (+ 37 ° C.) is terminated with four additional wash steps. Binding peroxidase activity that directly correlates with a number of binding anti-HIV antibodies is measured by adding H ₂ O ₂ / tetramethylbenzidine. After 30 minutes at room temperature, substrate conversion is stopped by addition of 0.5 M sulfuric acid. Absorption is measured at 450 nm.

Anti-HIV 1-positive samples and also particularly low-reactivity anti-HIV 1-positive samples, and anti-HIV-negative serum are studied in both reference systems and novel enzyme immunoassays.

Table 1 shows the results of the study (absorption unit).

Sample assignment Anti-HIV status Dilution Explanation Reference system Ⅰ New system Ⅰ Negative Control Course Positive Control 991/399111/399111/469111/469111 / 19BS 1-5BS 1-17BS 1-33BS 1-59 Voice positive voice positive voice positive positive voice negative voice negative voice 1: 40001: 1001: 2001: 501: 1001: 8000 High affinity Low affinity Low affinity Low affinity Low affinity High affinity 0.0981.5751.2110.5200.5280.3100.9060.0760.0770.0900.083 0.1030.4121.6530.8860.9270.4890.8770.0890.0780.0750.091

Significant differences appear in the signals produced in the two test systems, in particular the low affinity samples (eg 9111/46). The sensitivity for this sample is nearly twice that of Reference System I. Samples with high affinity for the coating antigen and already diluted to a high dilution rate in reference system I do not show an increased specific signal in novel system I. Anti-HIV-negative sera respond in a similar manner in both test systems.

Example 3: Use of New Reagents

3a) Enzyme Immunoassay to Detect HIV 1 Antibody (Reference System I)

Enzyme immunoassays (New System I) for detecting HIV 1 antibodies are as follows.

25 μl of sample buffer (0.3 M Tris / HCl, 1% albumin, 2% Tween 20, pH 7.2) was added at 37 ° C. with 100 μl of human serum in the well of a microtiter plate prepared as described in Example 1a above. Incubate for 30 minutes at. The plates were washed four times with 50 mM PBS, 0.1% Tween 20, followed by a conjugate prepared as described in Example 1b (0.1M Tris / HCl, 1 mM glycine, 0.2% albumin, 0.4% pluronine F64, human immunoglobulin For G, 125 μl of 1 mg / L biotinylated monoclonal antibody, diluted 1: 1000 at pH 8.1, as described in Example 1c) is pipetted into each well. The 30-minute incubation (+ 37 ° C.) is terminated with four additional wash steps. Binding peroxidase activity that directly correlates with a number of binding anti-HIV antibodies is measured by adding H ₂ O ₂ / tetramethylbenzidine. After 30 minutes at room temperature, substrate conversion is stopped by addition of 0.5 M sulfuric acid. Absorption is measured at 450 nm.

3b) Enzyme Immunoassay to Detect HIV 1 Antibody (New System II)

Enzyme immunoassays for detecting anti-HIV 1 antibodies are performed as follows.

25 μl of sample buffer (0.3 M Tris / HCl, 1% albumin, 2% Tween 20, pH 7.2) was added at 37 ° C. with 100 μl of human serum in the well of a microtiter plate prepared as described in Example 1a above. Incubate for 30 minutes at. The plate is washed four times with 50 mM PBS, 0.1% Tween 20 and 100 μl of dissociation buffer (5 mM glycine / HCl, pH 2.5) is added at RT for 30 minutes. A conjugate prepared as described in Example 1b (0.5M Tris / HCl, 1 mM glycine, 1% albumin, 2% fluronin F64, monoclonal antibody against human immunoglobulin G, which monoclonal antibody is carried out 25 μl of biotinylated 5 mg / L, 1: 200 dilution at pH 8.1, as described in Example 1c) is pipetted into each well without a wash step. The 30-minute incubation (+ 37 ° C.) is terminated with four additional wash steps. Binding peroxidase activity that directly correlates with a number of binding anti-HIV antibodies is measured by adding H ₂ O ₂ / tetramethylbenzidine. After 30 minutes at room temperature, substrate conversion is stopped by addition of 0.5 M sulfuric acid. Absorption is measured at 450 nm.

Anti-HIV 1-positive samples and also particularly low-reactivity anti-HIV 1-positive samples, and anti-HIV-negative serum are investigated in both the reference system and the novel enzyme immunoassay.

Table 2 shows the results of the study (absorption unit).

Sample assignment Anti-HIV status Dilution Explanation Reference system Ⅰ New system Ⅰ Negative Control Course Positive Control 991/399111/399111/469111/469111 / 19BS 1-5BS 1-17BS 1-33BS 1-59 Voice positive voice positive voice positive positive voice negative voice negative voice 1: 8001: 1001: 2001: 501: 1001: 8000 High affinity Low affinity Low affinity Low affinity Low affinity High affinity 0.1490.9971.8130.9880.6940.3410.9970.0980.0860.0890.084 0.1401.8342.5001.7140.8000.5021.8340.0900.0860.0800.079

Significant differences appear in the signals produced in the two test systems, in particular the low affinity samples (eg 9111/39). The sensitivity for this sample is nearly twice that of Reference System II. Samples with high affinity for the coating antigen and already diluted to a high dilution rate in the reference system II do not show an increased specific signal in the novel system II. Anti-HIV-negative sera respond in a similar manner in both test systems.

The present invention provides novel receptors R1, R2, R3, in order to overcome problems that may occur when immunoassay in a sample is measured by immunochemical methods, such as dissociation and reduced reactivity due to adverse reactions after immune complex formation. R4 is used to measure analytes in an immunochemically improved method.

Claims (11)

(1) an immunocomplex R1-A is prepared by immobilizing a sample which may contain the analyte to be measured, in contact with a receptor R1 immobilized on a solid phase and having a binding affinity for analyte A, (2) perform one or more washing steps, (3) add a labeled receptor R2 having at least one binding site with binding affinity for analyte A, and an unlabeled receptor R3 having at least one binding site with binding affinity for analyte A, wherein Receptors R2 and R3 can be added in any order, adding A, R1, R2, and R3 under initial dissociation conditions to induce degradation of the immune complexes, and then under non-relative conditions to allow recombination and renalization of the immune complexes. Preparing immobilized immune complexes containing (4) A method for measuring an analyte, including detecting in an appropriate manner the amount of label immobilized on the solid phase, which is a measure of the amount of analyte immobilized on the solid phase. 2. The method of claim 1, further comprising (1) immobilizing a receptor R4 having at least one binding site with binding affinity for receptor R3, and (2) said receptor R3 having affinity for analyte A A method for preparing an immune complex having at least one binding site and containing (3) A, R2 and R3 on the one hand and R1 and / or R4 on the other hand. (1) a sample, which may contain the analyte to be measured, is immobilized on a solid phase and contacted with a receptor R1 having a binding affinity for analyte A to prepare an immune complex R1-A, (2) perform one or more washing steps, (3) perform a dissociation step of inducing degradation of the immune complex R1-A, (4) add a labeled receptor R2 having one or more binding sites with binding affinity for analyte A, and an unlabeled receptor R3 with one or more binding sites with binding affinity for analyte A, wherein Receptors R2 and R3 can be added in any order under non-relative conditions to allow for the renalization of the immune complex, and analytes A and receptors R2 and R3 have one or more binding sites with binding affinity for receptor R3. An immobilized immune complex containing A, R2, R3 and R4 is prepared by contacting a secondary solid phase with R4 immobilized, (5) A method of measuring the analyte, including detecting the amount of label immobilized on the solid phase, which is a measure of the amount of analyte immobilized on the solid phase, in a suitable manner. The method of claim 1, wherein R1 and R3 are in each case an antibody or antibody fragment directed against the analyte and R2 is a labeled antibody directed against the analyte or labeled antibody fragment directed against the analyte. . 4. The labeled antibody according to claim 2, wherein R 1 and R 3 are in each case an antibody or antibody fragment directed against the analyte and R 2 is directed against a labeled antibody or analyte directed against the analyte. The antibody fragment and R4 is an antibody directed against R3 or an antibody fragment directed against R3. 6. The method of claim 5, wherein R3 is recognized as an epitope different from the epitope recognized as R1 or R2. The antibody of claim 1, wherein the analyte is an antibody, R1 is the antigen to which the analyte is directed, R2 is a labeled antigen to which the analyte is directed, and R3 is directed to the antibody or analyte directed to the analyte. The method is an antibody fragment. The method of claim 2 or 3, wherein the analyte is an antibody, R1 is the antigen to which the analyte is directed, R2 is a labeled antigen to which the analyte is directed, and R3 is the antibody or directed to the analyte. And an antibody fragment directed against R3 and R4 is an antibody directed against R3 or an antibody fragment directed against R3. The method of claim 8, wherein R3 involves an additional epitope that is not antibody-specific and R4 is directed to an additional epitope of R3 that is not antibody-specific. The method of claim 1, wherein the antibody against HIV is measured. A diagnostic agent for carrying out the method as claimed in claim 1.