MXPA99004995A - ANTIGEN-SPECIFIC IgG DETECTION - Google Patents

Abstract

The invention relates to a method for determining antigen-specific antibodies of immunoglobulin class G in the presence of immunoglobulins of class M in body fluids by incubation with at least two different receptors R 1 and R 2 and optionally additional receptors, wherein a bonding partner in monomer form is a substantial constituent of R 2. The invention also relates to a reagent for determining an antigen-specific antibody of immunoglobulin class G and the use of bonding partners in monomer form for determining an antigen-specific antibody of immunoglobulin class G.

Description

DETECTION OF SPECIFIC ANTIGENS FOR INMUNOGLOBU INA CLASS G DESCRIPTION OF THE INVENTION The invention relates to a method for the determination of antibodies specific for class G immunoglobulin antigen in body fluids by incubating the sample with at least two different receptors R. and R.sub.2 where both receptors are capable of specifically binding to the antibody, Rx binds or can bind to a solid phase and R2 presents a label, separate the solid phase from the liquid phase and measure the label where a binding partner in monomeric form which is specifically recognized by the antibody to be determined and a label is used as R2. In particular, the invention relates to a method for the specific detection of immunoglobulins of the IgG class in the presence of IgM immunoglobulin class. In response to the introduction of foreign substances, the immune system of the mammalian organism produces antibodies which are also called immunoglobulins. They defend it against foreign substances which are also called antigens. Immunoglobulins can be divided into five different classes. They differentiate between immunoglobulins of classes M, G, A, E and D. Each of REF .: 30318 these five classes of immunoglobulins differ in their composition of the heavy chain, which is called as chain μ,?, A , e and d. Each class of immunoglobulin has a different function in the organism. Immunoglobulins of class M appear with the first contact with the antigen, the so-called first immunization. However, the concentration of these immunoglobulins decreases rapidly as the infection progresses. Class G immunoglobulins are first formed slowly after a first immunization and are presented in large quantities when there is a second infection with the same antigen. Class A immunoglobulins are found on the mucous membrane surfaces of the body and are responsible for the defense processes in those places. Immunoglobulins of class E are the main responsible for allergic reactions. The exact function of class D immunoglobulins is unknown at this time. Individual classes of immunoglobulins occur at different concentrations in the blood. Thus, immunoglobulins of class G (IgG) are the main class in normal human serum, in which they constitute approximately 75% corresponding to the serum content of 8 to 18 mg / ml. The second immunoglobulin that occurs most frequently is IgA, which has an average serum concentration of 0.9 to 4.5 mg / ml. Immunoglobulins of class M are present in a concentration of 0.6 to 2.8 mg / ml, immunoglobulins of class D are present in a concentration of 0.003 to 0.4 mg / ml. The proportion of IgE antibodies is the lowest, occurring only at a concentration of 0.02 to 0.05 μg / ml in serum. For the differential diagnosis of many diseases, it is important to detect antibodies from one or several very particular classes of immunoglobulins which are specific for a particular antigen. A satisfactory diagnosis of viral, bacterial and parasitic infections can only be ensured by means of a class-specific antibody test or by excluding the presence of certain classes of immunoglobulins (for example the detection of IgG and IgA antibodies, but not detection). of IgM antibodies). This is particularly important for the differentiation between recent or acute infections and infections that have occurred previously as well as for clinical monitoring of the development of an infection. Specific detection of antibody class is especially important for HIV, hepatitis A, hepatitis B, toxoplasmosis, rubella and chlamydial infections. Specific detection of the class of antibodies specific for a particular antigen is also necessary for the determination of the titer of protective antibodies and for verifying the success of an immunization. Thus, from a diagnostic point of view, there is great interest in the detection especially of antibodies of the non-acute stages of infections such as IgG and IgA antibodies. Various methods have been described in the state of the art to detect antibodies of a particular class that are specific for an antigen. Therefore, antigen-specific antibodies of a particular kind are often detected by binding the specific antibodies to a solid phase coated with the specific antigen. Immunoglobulins (Ig) specific for the antigen which now bind to the solid phase are detected by antibody binding which specifically target human Ig of a certain kind to the Ig molecules to be detected. Antibodies directed to human Ig are provided with a label by means of which detection takes place. However, such a test procedure is only possible if all non-specific non-specific Ig are removed by washing prior to reaction with the class-specific labeled antibodies directed towards human Ig. Therefore, a single-stage test procedure often necessary for automated systems is not possible. In accordance with the method described in U.S. Patent No. 4,292,403, antibodies specific for antigen of a particular class of immunoglobulin are detected by immobilizing a class-specific antibody on a solid phase which binds sample antibodies to be determined, and they subsequently add specific antigens and binding to the antigen bound to an additional labeled antibody that is specific for the antigen. However, a disadvantage of this method is that all antibodies of the class to be determined must be bound to the class-specific immobilized antibody. The sample antibodies do not bind specifically to antigen. This can damage the sensitivity of the test since there may not be enough free binding sites for the antigen-specific antibody. Several washing steps are also necessary in this test procedure. This method does not allow a one-step test procedure. One possibility to carry out an antibody detection in a one-step test is provided by the so-called bridge test. The concept of bridge test is described in EP-AO 280 211. In this method, a first receptor which is capable of specific binding to the antibody to be determined binds to a solid phase such as, for example, an antigen The antibody to be determined binds to the antigen bound to the solid phase. In addition, an additional specific antigen is present in the reaction mixture which is provided with a label. The antibody is detected by means of the label. In this test all antibodies specific for antigen are detected and not only antibodies of a particular class. EP-A-0 307 149 and US Pat. No. 5,254,458 describe methods based on the principle of a bridge test for the detection of antibodies which are specifically directed towards an antigen. In this case, recombinantly produced peptides are used which are derived from a certain epitope of the antigen to bind the antibody to be detected. A peptide is immobilized to a solid phase. The sample antibody binds to the peptide. An additional labeled peptide is bound to the test antibody for detection. The recombinant peptides are expressed in different organisms in order to increase the specificity of the test. In addition, in this method antibodies of all its classes bind to the peptides. It is not possible, for example, to differentiate between IgG and IgM antibodies. EP-AO 386 713 describes a method for the detection of antibodies against HIV using two solid carriers in which antigens other than HIV are immobilized both in solid carriers which are each in contact with an aliquot of a sample as well as antigen of HIV labeling where the presence of antibodies is detected by a positive reaction in at least one of the tests.

The recombinantly produced polypeptides are described as HIV antigens. A method based on Western blotting is described in EP-A-0 627 625 in which antibodies for HIV can also be detected by means of recombinant proteins or synthetic peptides. However, both methods do not allow specific class detection of antigen-specific antibodies. The previous methods do not allow a specific antibody to antigen of a certain immunoglobulin class to be detected in a single-step method. Immunological methods of detection known from the state of the art based on the concept of bridging test in which a labeled antigen is used and an antigen capable of binding to a solid phase, actually allow a single-step test. However, until now it has not been possible to jointly detect antibodies of the IgG and IgM classes using this simple principle. Therefore, the aim is to provide an improved method for the detection of antibodies of a non-acute infection directed towards a specific antigen, that is, in particular of the IgG class. At the same time, the method should ensure that IgM antibodies of the same specificity are not detected. This method preferably consists of a one-step test principle in order to be advantageously used in automated systems.

This objective is obtained by the method according to the invention for the determination of a specific antibody for antigen of the class of immunoglobulin G by incubating the sample with at least two different receptors R2 and R2 wherein both receptors are capable of specifically binding to the Rx antibody binds or can bind to a solid phase and R2 presents a label which is characterized in that R2 is used as a conjugate of a binding partner in monomeric form that is specifically recognized with the antibody to be determined, and a label. The method according to the invention allows the determination of antibodies specific for antigen of the class of immunoglobulin G in samples in which antibodies of the IgM class of the same antigen specificity are present. The IgA, IgD and IgE antibodies present in the sample which have the same specificity as the IgG antibodies to be detected, present in much lower concentrations than the IgG antibodies. In particular, the IgD and IgE classes are present in concentrations that are several orders of magnitude lower than the IgG concentration, so that their reactivity in the detection method does not change or hardly changes the measured result. IgA antibodies are present in concentrations corresponding to approximately 10% of the total IgG content. Therefore, IgA antibodies can also be determined presumably in this method. Since the main purpose of the method is to detect antibodies from non-acute infection, the joint detection of IgG and IgA antibodies, both of which are non-acute, is not critical. Since IgG antibodies are the main class of immunoglobulin in a non-acute infection, the term IgG detection is used in the following. It is essential that IgM antibodies of the same antigen specificity should not be detected, which only occur in large quantities in an acute infection. Surprisingly it has become evident that the use according to the invention of binding partners in monomeric form in a bridge test based on the double antigen test principle allows only antibodies of the IgG class to be detected which are specifically targeted towards a particular antigen. Antibodies in the IgM class of the same specificity which are present in the same sample surprisingly do not react or only to a negligibly weak degree with monomeric peptides and therefore do not interfere with the detection of IgG. The term "negligibly weak" means that the antigen binding sites of the IgM antibodies are not detectably linked by the binding partners in monomeric form. This is probably due to the much lower affinity of the pentameric IgM antibodies for monomeric epitopes compared to the IgG antibodies present in the form of individual molecules. Thus, a successive test procedure is not absolutely necessary to separate the antibodies IgM in the method according to the invention, since these do not interfere. A particular advantage of the method therefore is the simplicity of the test procedure. In addition to the so-called wet tests in which the test reagents are present in a liquid phase, all standard dry test formats which are suitable for the detection of proteins or antibodies can also be used. In these dry tests or test strips, as described for example in EP-A-0 186 799, the test components are applied to a carrier. Therefore, if the method according to the invention is carried out in a test strip format, a washing step is not necessary. However, the method according to the invention is preferably carried out as a wet test. It is possible to incubate all the receptors and the sample together, and carry out the method in a single step. This optionally requires only one wash step after incubation.

Normally two different receivers Rx and R2 are used to carry out the method according to the invention.

If a wet test is used, the R2 receptor is present in a liquid phase. R-_ may be present in a liquid phase or already bound to the solid phase. If R-_ and R2 are present in a liquid phase, they are preferably at the same concentration. The concentration ratios of Ri.-Rs of 0. 5: 1.0 to 1.0: 5.0 have proven to be adequate. The optimum concentration ratios can be easily determined by a person familiar with the art. If a receptor capable of binding to a solid phase but which has not yet been bound to the solid phase is used as Rx, the sample is subsequently incubated with the Rx and R2 receptors. In this process, the sample antibody binds to R. and R2. This incubation can be carried out in the presence of the solid phase. A complex is formed in this process comprising the solid phase-R1-sample-antibody-R2. Subsequently the solid phase is separated from the liquid phase, the solid phase optionally washed and the label of R2 is measured. The label is usually measured in the solid phase, however it can also be determined in the liquid phase. If the incubation of the sample with Rx and R2 is carried out in the absence of the solid phase, then the entire test mixture must subsequently be contacted with the solid phase, and washing is optionally carried out and measured the label. If the R. receptor is already in solid phase bound form, then the sample and the R2 receptor are added to the R receptor bound to the solid phase and incubated together. The additional procedure corresponds to the process established before. However, it is also possible to carry out the method according to the invention in several stages. In this case it is expedient to first incubate the sample with the Rx and R2 receivers. The complex that is formed of R-_, R2 and antibody to be determined is subsequently incubated with other receptors so this can be carried out in several stages. The additional test procedure corresponds to the method described previously. The receptor R2 is a conjugate of a binding partner in monomeric form and a label. The binding partners according to the invention in monomeric form contain exactly one epitope region or only one binding site for the antibody to be determined, ie, a structure that immunologically reacts specifically with the IgG antibody that is will determine. The monomeric structure of the binding partner is important to ensure that only IgG antibodies specific for antigen to be detected bind to the binding partner in monomeric form and not the interfering IgM antibodies of the same specificity. The epitope region, for example, can be derived from an antigen or an anti-idiotype antibody. The epitope regions, in the case of the binding partners in monomeric form, they can also be derived from sugar structures and / or lipids or structures combined with peptide, lipid and / or sugar components. All structures that can be derived from an epitope region which have a binding site to which the antibody of the IgG class to be detected in the presence of IgM antibodies of the same specificity are specifically linked can be used. The only prerequisite for the binding site, ie, for the binding partners used in monomeric form is that the specific capacity for binding to IgG is retained. This condition also applies to the case where sugar or lipid structures are present at the binding site. In accordance with the invention, it is also possible to use binding partners in monomeric form which flank or overlap the binding site to which the IgG antibody to be detected specifically binds. Therefore, it is also possible to detect IgG antibodies that provide a cross reaction whose binding sites overlap with the epitope to be detected. Therefore, a mixture of binding partners in monomeric form is preferably used to detect antigen-specific IgG antibodies. The peptides are preferably used as binding partners in monomeric form. In the case of a protein as an analyte, a binding site is understood as a peptide, the sequence of which is part of the protein sequence of a protein antigen and to which an antibody directed to this protein specifically binds, the which, in the case of the present invention is an IgG antibody. In addition to these peptides, it is also understood that the binding site includes peptides with amino acid sequences which have an essentially equivalent specificity and / or binding affinity to the IgG antibody to be detected as the peptides mentioned above. These peptides are preferably derived from the aforementioned peptides by substitution, deletion or insertion of individual amino acid residues. All peptides can be used which have a binding site to which the IgG class antibody to be determined specifically binds, even in the presence of IgM antibodies of the same specificity. The only prerequisite for the binding site, ie for the peptide used is that its ability to specifically bind to IgG is retained. A binding site is understood as a peptide whose sequence is part of a protein sequence or a protein antigen (analyte) and to which an antibody directed against this protein specifically binds, which in the present invention is an IgG antibody. In addition to these peptides, it is also understood that the binding site includes peptides with amino acid sequences which have specificity and / or essentially equivalent binding affinity to the IgG antibody to be determined as the peptides mentioned above. These peptides can preferably be derived from the aforementioned peptides by substitution, deletion or insertion of individual amino acid residues. The peptides according to the invention which correspond to a specific binding site are also understood to include peptide derivatives in which one or more amino acids have been derivatized (have formed derivatives) by a chemical reaction. Examples of peptide derivatives according to the invention are in particular those molecules in which the main structure and / or the groups of reactive side amino acids, for example free amino groups, free carboxyl groups and / or free hydroxyl groups, they have been derivatized. Specific examples of derivatives of amino groups are sulfonamides or carboxamides, thiurethane derivatives and ammonium salts, for example hydrochloride. Derivatives of carboxyl groups are salts, esters and amides. Examples of derivatives of the hydroxyl group are O-acyl derivatives or O-alkyl derivatives. The peptides are preferably produced by chemical synthesis according to methods known to those familiar with the art and need not be elucidated especially here. At first, the peptides can also be produced by means of recombinant methods. However, longer polypeptides often tend to dimerize or polymerize, so that the peptides are preferably produced by chemical synthesis in order to ensure the monomeric properties. In addition, the term peptide derivative also encompasses such peptides in which one or more amino acids have been replaced by homologs of naturally occurring or non-naturally occurring amino acids of the 20 amino acids ("standard." Examples of such homologues are 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine, ornithine, β-alanine and 4-aminobutyric The peptide derivatives must have a specificity or / and essentially equivalent binding affinity to the IgG antibodies to be determined as the peptides to which they are derived The peptides according to the invention which correspond to a specific binding site are also referred to as peptidomimetic substances termed peptide mimetics in the following which have essentially a specificity and / or equivalent affinity of binding to the IgG antibodies that are to be determined as the peptides mentioned before or after Peptide mimics Peptidomimetics are compounds which can replace the peptides with respect to their interaction with the antibody to be determined and may have a higher stability compared to native peptides, in particular towards proteinases and peptidases. Methods for the production of peptidomimetics are described in Giannis and Kolter, "Ange. Chem." 105 (1993), 1303-1326 and Lee et al., Bull. Chem. Soc. Jpn. 66 (1993), 2006-2010. The length of a binding site, ie, the length of a monomeric peptide according to the invention is usually at least 4 amino acids. Preferably, the length is between 4 and 20, 6 and 15 or particularly preferably between 9 and 12 amino acids. In the case of peptidomimetics or peptide derivatives, an analogous length is necessary with respect to the size of the molecule. The monomeric peptides according to the invention as a binding partner in a monomeric form contain the epitope to which the IgG antibody to be determined specifically binds. However, additional flanking sequences which no longer correspond to the specific epitope may be present at the N-terminus and / or at the C-terminus of the peptide. In addition, it is possible for the peptide to be provided with spacer groups known to a person familiar with the art. The only prerequisite is that the peptide as a binding partner in a monomeric form is actually present as a monomer and retains its ability to bind the IgG antibodies to be determined. An additional component of the R2 receiver is the label. Preferably a directly detectable substance is used as a label, for example, a chemiluminescent, fluorescent or radioactive substance or a metallic sol, latex or gold particle. Enzymes or other biological molecules are also preferred as the label such as for example haptens. Digoxigenin is a particularly preferred label among haptens. All processes for labeling are known to a person familiar with the art and do not need to be further elucidated here. The label is detected directly in a well-known manner by measuring the chemiluminescent, fluorescent or radioactive substance or the metallic sol, in latex, or gold particle or by measuring the substrate converted by the enzyme. The label can also be detected indirectly. In this case, an additional receiver which itself in turn is coupled to a signal generating group specifically binds to the label of R2 such as a hapten for example as digoxigenin. The signal generator group, for example a chemiluminescent, fluorescent or radioactive substance or an enzyme or gold particle, is detected by familiar methods to a person familiar with the art. For example, an antibody or an antibody fragment can be used as the additional receptor which binds specifically to the tag of R2. If this indirect detection of the label is used then the label R2 preferably digoxigenin or another hapten and the detection is carried out via an antibody coupled to peroxidase which is directed towards digoxigenin or towards the hapten. An essential component of the Rx receptor is a binding partner which is capable of specific binding to the IgG antibody to be determined. The Rx receptor can bind directly to the solid phase or is capable of binding to the solid phase. A binding partner in a monomeric form as in the R2 receptor can be used as the binding partner which is capable of specific binding to the IgG antibody to be determined. However, it is also possible to use binding partners which are not present in a monomeric form, that is, the binding partner can have more than one epitope or binding site. It is important that the ability of the binding partner to bind specifically to the IgG antibodies to be determined be retained. However, binding partners in a particularly preferred monomeric form and peptides are also used for the R receptor. The peptides contained in R. are produced by the same methods as the peptides for R2. The antibody-specific binding partners or peptides contained in the Rx and R2 receptors may be identical or different, but both must be capable of simultaneously binding to the IgG antibody to be determined. Rx can be linked either directly to the solid phase. The direct binding of Rx to the solid phase is obtained by methods known to a person familiar with the art. R ^ can also be linked directly to the solid phase by means of a specific binding system. In this case, R is a conjugate which is constituted by a peptide as elucidated above and a reaction partner of a specific binding system. In this case, a specific joining system is understood as two associates which can react specifically together. In this case, the binding capacity can be based on an immunological reaction or on another specific reaction. Preferably a combination of biotin and avidin or of biotin and espreptavidin is used as a specific binding system. Other preferred combinations are biotin and antibiotin, hapten and antihapten, Fc fragment of an antibody and antibody against this Fc fragment or carbohydrate and lectin. One of the reaction partners of this specifically univable pair is then a part of the conjugate that forms the Rx receptor. The other reaction partner of the specific binding system is present, then, in a solid phase. The other reaction partner of the specific binding system can be attached to an insoluble carrier material by conventional methods known to one of ordinary skill in the art. In this case, a covalent as well as an absorptive bond is suitable. Solid phases which are particularly suitable are test tubes or microtiter plates made of polystyrene or similar plastics, the inner surfaces of which are coated with the reaction partner of the specific binding system. Particulate substances such as latex particles, molecular sieve materials, glass spheres, plastic tubes, etc. are also suitable, and are particularly preferred. Porous layered carriers can also be used as the carrier. In a preferred embodiment of the method according to the invention, a conjugate consisting of a binding partner in a monomeric form and a reaction partner of the specific binding system is used as R. In this preferred embodiment the R and R2 receptors are incubated together as well as the sample which contains the IgG antibody to be determined. In this process, the peptide components of the R1 and R2 receptors react specifically with the IgG antibodies to be determined. This complex consisting of Rl t sample antibody and R2 is bound to the solid phase which is coated with another reaction partner of the specific binding system by means of the reaction partner of the specific binding system which is a component of R1. As a result, the entire complex of Rlf sample antibody and R2 is bound to the solid phase. After the solid phase has been separated from the liquid phase and the solid phase is optionally washed, the R2 label is detected by methods known to a person familiar with the art. This test procedure allows IgG antibodies to be detected specifically in the presence of IgM antibodies of the same specificity. In a further preferred embodiment of the method according to the invention, an additional receiver is used in addition to the Rx and R2 receptors. In this test procedure, a conjugate consisting of a binding partner in monomeric form and a reaction partner of a specific binding system such as, for example, biotin is used as Ri. For this, the R? Receptors are incubated together? and R2 as well as the sample which contains the IgG antibody to be determined. In this process, the peptide components of the R and R2 receptors react specifically with the IgG antibodies to be determined. The binding to the solid phase which is coated with the other reaction partner of the specific binding system (for example with streptavidin) is obtained by means of a reaction partner of a specific binding system which is a component of R1. As a result, the entire complex consisting of R, R2 and the sample antibody bind to the solid phase. After separating the solid phase from the liquid phase and optionally washing the solid phase, the complex bound to the solid phase is incubated with an additional receptor which specifically recognizes the R2 tag. The additional receiver is coupled to a signal generating group such as an enzyme. After an optional washing step, the sample antibody is detected via the signal generating group, in this case by the substrate converted by the enzyme. If this test procedure is used, digoxigenin is preferably used as the R2 tag. In this case, the additional receptor is constituted by an antibody or a fragment antibody directed towards digoxigenin and the enzyme peroxidase. In this test procedure, incubation of the sample with R-1 and R 2 and the additional receptor can also be carried out concurrently. This test procedure is also very suitable for an application to automated systems but requires two or more washing steps. A major advantage of this test procedure becomes apparent if it is intended to detect several antigen-specific antibodies such as, for example, HIV antibodies against gp41, pl7, etc. In such a case, the additional receiver can be used as a universal label since this additional receiver specifically recognizes the R2 tag. All biological liquids known to a person familiar with the art can be used as samples. Preferably, body fluids such as whole blood, blood serum, blood plasma, urine, saliva, etc., are used as the sample. In addition to the sample, the solid phase and the receptors mentioned above, other additives may be present in the test mixtures which may be required on the basis of the application, such as buffers, salts, detergents, protein additives such as BSA. The necessary additives are known to a person familiar with the art or can be determined by the same person in a simple manner. In order to ensure that IgM antibodies or rheumatoid factors do not interfere with the detection of antigen-specific IgG, it is possible to optionally use additional measures for interference reduction. These may include, for example, the use of reducing substances such as dithiothreitol (DTT), dithioerythritol (DTE) or β-mercaptoethanol in the approach described in EP-B-0 341 439. In addition, antibodies may optionally be used against Fd to eliminate interference by rheumatoid factors. Such a concept is described in WO 96/14338. The various measures to reduce interference can be used individually or in any combination. A further subject matter of the invention is a reagent for the determination of an antigen-specific antibody of the class of immunoglobulin G which, in addition to the usual test additives for immunoassays such as buffers, salts, detergents, etc., contains a receptor R2 capable of binding to the antibody to be determined which is constituted by a binding partner in a monomeric form, and a label. A subject matter of the invention is also a reagent for the determination of an antigen-specific antibody of the class of immunoglobulin G which, in addition to the usual test additives for immunoassays, contains two receptors Rx and R2 capable of binding to the antibody that is going to be determined, of which R? it is capable of binding to a solid phase and R2 carries a label wherein an essential component of the receptor R2 is a binding partner in a monomeric form. A further subject matter of the invention is also a reagent for the determination of an antibody specific for antigen of the class of immunoglobulin G which, in addition to the usual test additives for immunoassays, contains two receptors Rx and R2 capable of binding to the antibody which is to be determined, of which Rx is capable of binding to a solid phase and R2 carries a label wherein an essential component of both receptors is a binding partner, in a monomeric form. In addition, a subject matter of the present invention is the use of binding partners in a monomeric form to determine the antigen-specific antibody of the immunoglobulin class G. The invention is elucidated by the following examples.

Examples 1. Reactivity with < HIV 2 > MAB (IgG and IgM) when monomeric and multimeric epitopes are used Description of the test procedure: Antigens labeled with biotin and labeled with monomeric digoxigenin (test A) or multimeric (test B) (HIV 2) react with sample antibodies, * and a solid phase coated with streptavidin (incubation at 25 ° C or 37 ° C ca. 60 to 180 min, in this example: 120 min, 25 ° C). After a washing step, the immune complex bound to the wall reacts with an anti-digoxigein-peroxidase conjugate (incubation at 25 ° C or 37 ° C, ca. 30 to 120 min, in this example: 60 min, 25 ° C) . After an additional washing step, the immune complex labeled with peroxidase conjugate is detected by a substrate reaction (incubation of the conjugate, 60 min at 25 ° C). In general, incubation of the conjugate can be carried out at 25 ° C or 37 ° C for ca. 30 to 120 min. The reaction steps (in addition to the substrate reaction) take place in a Tris-HCl buffer (pH 7.5, 50 to 150 mM, in this example 100 mM) containing ca. 0.05 to 0.4% detergent (here, 0.2% polidocanol) and ca. 0.5% protein / protein-derived additive (here lactalbumin peptone and BSA, among others). In this case, the sample antibodies are mouse monoclonal antibodies (IgM and IgG) against an HIV 2 epitope diluted at ca. 2-20 g / μl of human serum negative against HIV.

Table 1: Comparison test results of tests A and B: Absorbances in mA The use of specific binding partners for HIV 2 in A monomeric form allows the specific detection of IgG antibodies against HIV 2. Antibodies of the IgM class of the same antigen specificity are not recognized (test A). When specific binding partners for HIV 2 are used in a multimeric form, it is not possible to differentiate between IgG and IgM (test B). 2. Reactivity with serum antibodies from a seroconversion of HIV2 in a chimpanzee Experimental procedure as in example 1 Table 2: Test results of a seroconversion of a chimpanzee with tests A and B: Absorbances in mA The use of the binding partners in a monomeric form allows seroconversion after the HIV2 infection has been detected. The advantage of test A is particularly evident at week 3 after infection: Only IgM antibodies are present which are not detected by the binding partners in a monomeric form. Test A only shows a positive signal after IgG antibodies appear. Test B, which uses multimeric epitopes, is not capable of differentiating between IgM and IgG with the same specificity. 3. Reactivity with serum antibodies from a seroneversion of HIV1 from a patient Experimental procedure as in example 1, but HIV 1 antigens.

Table 3. Test results of an HIV1 seroconversion with tests A and B Adsorbances in mA Test A allows reliable detection of IgG antibodies also in the case of infection with HIVl (analogues to HIV2 according to example 2). 4. Detection of rubella IgG are peptides containing monomeric epitopes Description of the test procedure: Rubella specific IgG is detected by means of associates according to the invention in a monomeric form on an Elecsys ™ 2010 instrument from Boehringer Mannheim GmbH, Germany, according to the manufacturer's instructions. The following reagents are used: Reagent Rl: antigen cyclic peptide rubella, biotinylated Tris buffer, pH, 7.5, 0.2% Myrij, 0.2% BSA, 0.1% R-IgG Reagent Rl: antigen cyclic peptide rubella, ruthenized Tris buffer, pH 7.5, 0.2% Myrij, 0.2% BSA, 0.1% R-IgG. _ The samples of human serum were used as samples.

The test is carried out in the following stages: 1. 30 μl of sample + 65 μl of Rx + 65 μl of R2 2. incubation at 37 ° C, 9 min 3. addition of 40 μl of magnetic beads coated with SA 4. incubation at 37 ° C, 9 min 5. reaction of detection: measuring the electrochemiluminescent signal Table 4: Results Only samples containing antigen-specific IgG are recognized as positive. Samples which only contain antigen-specific IgM (No. 1.3 and 1.4) are not recognized according to the invention as positive.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention is the conventional one for the manufacture of the objects or products to which it refers.

Claims (9)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for the determination of an antigen-specific antibody of the immunoglobulin G class by incubating the sample with at least two different receptors R1 and R2 in which both receptors are capable of specifically binding to the antibody, R is bound to a phase solid or can bind to a solid phase, and R2 presents a label, wherein a conjugate of a binding partner in monomeric form which is specifically recognized by the antibody to be determined and a label is used as R

2. 2. The method according to claim 1, characterized in that an essential component of Rx is a binding partner in monomeric form.

3. The method according to claim 1 or 2, characterized in that biotin / avidin is used; biotin / streptavidin; biotin / antibiotin; hapten / antihapten; Fc fragment or an antibody / antibody against this fragment of Fc; carbohydrate / lectin, as the specific binding system to bind R to the solid phase. The method according to one of claims 1 to 3, characterized in that the receptor R2 is labeled with a chemiluminescent, fluorescent or radioactive substance, an enzyme or another biological molecule. The method according to one of claims 1 to 4, characterized in that the sample is incubated simultaneously with R ± and R2. The method according to one of claims 1 to 5, characterized in that the test mixture is incubated with an additional receptor which specifically binds to the label of the receptor R2, wherein the additional receptor is a conjugate of a receptor. which is specific to the label of R2 and a label, and the label is determined subsequently. 7. The method of. according to one of claims 1 to 6, characterized in that the amount of binding partner in monomeric form in R-γ that is specifically recognized by the antibody to be determined is greater than or equal to the amount of binding partners in monomeric form in R2 which is specifically recognized by the antibody to be determined. 8. A reagent for the determination of an antigen-specific antibody of the class immunoglobulin G according to one of the previous claims, characterized in that in addition to the usual test additives for immunoassays, it contains a receptor R2 capable of binding to the antibody that is will determine which is constituted by a binding partner in monomeric form and a label. 9. The use of the binding partners in monomeric form, characterized in that they are used for the determination of an antigen-specific antibody of the class immunoglobulin G.