MXPA99004991A - ANTIGEN-SPECIFIC IgM DETECTION - Google Patents

Abstract

The invention relates to a method for determining antigen-specific antibodies of immunoglobulin class M in the presence of immunoglobulins of class G and/or rheumatoid factor 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 polymer form is a substantial constituent of R 2 and the disturbing effects of IgM antibodies with the same antigen specificity are suppressed by bonding partners in monomer form. The invention also relates to a reagent for determining an antigen-specific antibody of immunoglobulin class M and the use of bonding partners in monomer form in suppressing the disturbing effects of IgG antibodies upon determination of antigen-specific IgM antibodies.

Description

DETECTION OF SPECIFIC ANTIGENS FOR IMMUNOGLOBULIN M CLASS DESCRIPTION OF THE INVENTION The invention relates to a method for the determination of antigen-specific antibodies of the immunoglobulin class in body fluids by incubating the sample with at least two different receptors R and R2 wherein both receptors are capable of specifically binding to the antibody, Rx it binds or can bind to a solid phase, and R2 carries a label, where an essential component of Ki and optionally also of R2, is a binding partner in polymeric form which is specifically recognized by the antibody that is going to determining and using binding partners of the same specificity in monomeric form to reduce interference by IgG antibodies. In particular, the invention relates to a method for the specific detection of immunoglobulins of the IgM class in the presence of immunoglobulins of the IgG class and interference factors such as retinoid factors. In response to the introduction of foreign substances, the immune system of a mammalian organism produces antibodies which are also called immunoglobulins. They defend it against foreign substances which are also called antigen. The immunoglobulins are REF .: 30319 can be divided into five different classes. One difference between immunoglobulin of classes M, G, A, E and D. Each of these five classes of immunoglobulin differs in the composition of the heavy chain which is referred to as a chain, μ?, A, £ Y d . Each class of immunoglobulin has a different function in the organism. Immunoglobulins of class M appear after the first contact with the antigen, the so-called primary immunization. However, the concentration of these immunoglobulins decreases rapidly as the infection progresses. The immunoglobulins of class G first form slowly after primary immunization and occur in large quantities when there is a second infection with the same antigen. Immunoglobulins of class A are found on the mucous membrane surfaces of organisms and are responsible for the defense processes in that place. Immunoglobulins of class E are responsible mainly for allergic reactions. The exact function of immunoglobulins of class D is unknown until now. Individual classes of immunoglobulin occur in very different concentrations in the blood. Thus, immunoglobulins of class G (IgG) are the main class in normal human serum which 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 at a concentration of 0.6 to 2.8 mg / ml, immunoglobulins of class D are present at a concentration of 0.003 to 0.4 mg / ml. The proportion of IgE antibodies is the lowest and only occurs 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 of one or several very particular classes of immunoglobulin which are specific for a particular antigen. A satisfactory diagnosis of viral, bacterial and parasitic infections can only be produced by means of a class-specific antibody test or by excluding the presence of certain classes of immunoglobulin (eg detection of IgA IgG antibodies, but no detection of antibodies). IgM). This is particularly important to differentiate between recent or acute infections and infections that have occurred previously as well as for clinical monitoring of the use of an infection. Specific detection of antibody class is especially important for HIV, hepatitis A, hepatitis B, toxoplasmosis, rubella and chlamydial infections. Class-specific detection of antibodies specific for a particular antigen is also necessary when determining the titer of protective antibodies and for verifying the success of an immunization. For the diagnosis of recent acute infections, it is of particular interest to detect antibodies of the IgM class which are specific for an antigen. However, various interference factors such as for example the presence of IgG antibodies of the same specificity often interfere with a detection of antigen-specific IgM 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 frequently detected by binding of 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 binding antibodies to which they specifically target human Ig of a certain kind to the Ig molecules to be detected. Antibodies directed towards human Ig are provided with a label by means of which detection takes place. However, such a test procedure is only possible if such nonspecific non-bound Ig is removed from the wash prior to reaction with the class-specific labeled antibodies directed towards human Ig. Therefore, a one-step test procedure as is often required for automated systems is not possible. In addition, antibodies of all classes which are specific for the antigen bind to the solid phase of the first stage. If the antigen coating of the solid phase is not sufficiently high, competition reactions of various kinds of antibodies for antigen binding may occur. This can damage the weakness of the test. One possibility to carry out an antibody detection in a one-step test is provided by what is called a bridge test. The concept of bridge test is described in EP-A-0 280 211. In this method, a first receptor is attached to a solid phase such as for example an antigen which is capable of specific binding to the antibody that is going away. to determinate. The antibody to be determined binds to the antigen bound to the solid phase. In addition, an additional specific antigen is present in the test mixture which is provided with a label. The antibody is detected by means of the label. However, in this test all antigen-specific antibodies are detected and not only the antibodies of a particular class. Additional interference when antigen-specific IgM antibodies are determined is caused by rheumatoid factors. The rheumatoid factors themselves are usually antibodies of the IgM class which generally have a high affinity for the Fe regions of the IgG antibodies. As a result, the rheumatoid factors apparently present IgG antibodies so that the rheumatoid factors bind in an immunoassay for IgM-specific antibodies. If rheumatoid factors have found IgG molecules with the specificity to be detected, this may result in false-positive measurement results. This problem in the specific detection of class of antigen-specific antibodies is the subject matter of DE 33 03 793. This describes a method for the detection of an antigen-specific antibody of a certain Ig class ("IgX") in which Eliminates interference by rheumatoid factors by adding antibodies against IgG. In the specific antigen method, such as a viral antigen, it is applied to a solid carrier. The viral antigen bound to the solid phase is contacted with the sample. In the next stage, the unbound sample is removed and the solid-phase bound complex of the IgX-antigen is detected with an antibody against IgX. In order to avoid interference by rheumatoid factors especially in IgM tests, the sample is treated with antibodies against IgG before the test. IgG antibodies form complexes in this way and therefore are no longer available for binding to rheumatoid factors so that rheumatoid factors are not able to bind antigen-specific IgG molecules which in turn can result in false-positive results . However, all IgG antibodies bind regardless of their specificity. The precipitation of IgG antibodies that interfere with antibodies against IgG can lead to unwanted precipitates and turbidity which can have adverse effects on the entire test. In addition, a pretreatment of the sample with antibodies against IgG is complicated. A further method for eliminating interference by antibodies of other classes with the same specificity is described in WO 96/14337. In this case, antibodies or antibody fragments which react specifically with the Fd section of the heavy chain of IgG are used to eliminate interference by IgG antibodies. As a result, the antigen binding capacity of IgGs is masked so strongly that they are no longer able to recognize specific antigens. A similar concept is described in WO 96/14338. In this case, antibodies against Fd or fragments thereof are used as a reagent to eliminate interference in order to reduce interference by rheumatoid factors. However, the reduction of interference by a highly specific reagent against Fd is complicated and expensive. The methods known in the prior art do not allow the detection of an antigen-specific antibody of the IgM immunoglobulin class in a one-step method without having to add elaborate and expensive reagents that eliminate the interference and / or several specific antibodies. Immunological methods of detection known from the state of the art based on the concept of bridging test in which a labeled antigen and an antigen capable of binding to a solid phase are used actually allow for a one-step test. However, until now it has only 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 Ig class antibodies directed against a specific antigen. This method does not require elaborate and expensive reagents that eliminate interference and preferably consist of the one-step test principle in order to be advantageously used in automated systems. This object is obtained by the method according to the invention for the determination of an antigen-specific antibody of the immunoglobulin M class by incubating the sample with at least two different receptors, R? and R2 wherein both receptors are capable of specifically binding to the antibody, Rt binds or can bind to a solid phase, and R2 has a label, wherein an essential component of R is a binding partner in polymeric form which is specifically recognized by the antibody to be determined, and interference by IgG molecules of the same specificity present in the sample is eliminated by adding binding partners in monomeric form. The IgA, IgD and IgE antibodies present in the sample which have the same specificity as the IgM antibodies to be detected are presented in very low concentrations compared to the IgG antibodies so that interference of the IgA classes can not be expected. , IgD and IgE. The IgA, IgD and IgE antibody classes are - like the IgG molecules and in contrast to the IgM antibodies that are present as a pentamer antibodies which are present in the form of single molecules and each has two binding sites for the antigen. Thus, due to the structural similarity of IgG, IgA, IgD and IgE antibodies, the interference by IgD, IgA and IgE antibodies is also probably reduced in addition to that caused by IgG antibodies by the method for detection of IgM specified antigen described in the next . The method according to the invention allows the determination of antigen-specific antibodies of the immunoglobulin class in samples in which antibodies of the IgG class with the same antigen specificity are present. In addition, the method according to the invention can be carried out in the presence of rheumatoid factors. An elaborate pretreatment of the sample is not necessary. Surprisingly it has been noted that the use according to the invention of binding partners in a monomeric form in an immunoassay for the detection of antigen-specific IgM antibodies allows the effective elimination of interference caused by IgG antibodies with the same antigen specificity. . In this method, the binding partners in a monomeric form bind specifically to the antigen binding sites of the IgG antibodies. The IgM antibodies to be detected with the same specificity which are present in the same sample surprisingly do not react or only do so to a negligible weak degree with the binding partners in a monomeric form. The term "negligibly weak" means that the antigen binding sites of the IgM antibodies are not blocked by 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 which have a substantially higher affinity for monomeric epitopes. This means that, despite the presence of binding partners in a monomeric form, the sensitivity of the IgM test is not impaired. The I antibodies masked by the binding partners in a monomeric form do not interfere with the IgM test. It has also been surprisingly found that interference by rheumatoid factors having bound IgG antibodies can also be effectively eliminated by the binding partners in monomeric form which bind to the antigen binding sites of the IgGs. Since the antigen binding sites of IgG antibodies are blocked, antigen-specific Ig antibodies can be detected without prior separation of IgG antibodies or rheumatoid factors. The binding partners used in monomeric form are not capable of activating an agglutination reaction of masked IgG antibodies or rheumatoid factors. This prevents undesirable turbidities due to precipitates which may adversely affect the entire test procedure. Therefore, a successive test procedure for separating the IgG antibodies is not absolutely necessary in the method according to the invention since these do not interfere. Therefore, a particular advantage of the method 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 binding partners in a monomeric form together with the sample and carry out the method in one step. This optionally requires only one washing step after incubation. Normally, two different receptors R1 and R2 and the binding partners are used in a monomeric form to carry out the method according to the invention. If a wet test is used, the R2 receptor is present in a liquid phase. Rx may be present in a liquid phase or already bound to the solid phase. The binding partners in a monomeric form are preferably present in the liquid phase. If it is used as R? a receptor capable of binding to a solid phase but which has not yet been bound to the solid phase, the sample is then incubated together with the R? and R2 and l ° e binding partners in a monomeric form.

In this process, the antibody in the sample binds to R2 and R2. This incubation can be carried out in the presence of the solid phase. In the process a complex is formed which is constituted by solid phase-Rlf antibody-sample-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 solid phase but it can also be determined in liquid phase. If the incubation of the sample is carried out with Rx and R2 and as well as the binding partners in monomeric form in the absence of the solid phase, then the entire test mixture can subsequently be contacted with the solid phase, and the washing is optionally carried out and the label is measured. If the RL 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. In this test procedure, the sample is preferably preincubated with the binding partners in a monomeric form and R2 is added before the test mixture to the R1 receptor bound to the solid phase. The additional procedure corresponds to the methods indicated above. It is also possible to carry out the method according to the invention in several stages. In this case, the sample is preferably incubated with the binding partners in a monomeric form and then with the Rx and R2 receptors. The test sample can subsequently be incubated with other receptors where this can be carried out in several stages. The additional test procedure corresponds to the method described previously. An important component of Rx is a binding partner in a polymeric form which is specifically recognized by the IgM antibodies to be determined, which are also referred to as a polyhapten. A binding partner in a polymeric form according to the invention is understood as structures in which are coupled several equivalent preferably identical or similar epitope regions, which react specifically with the antibody to be determined. The term "similar" or "equivalent" means that the present structures of the binding partner in a polymeric form do not necessarily have to be all identical. The only condition is that the IgM antibodies to be determined bind specifically to these epitope regions. The epitope region can be derived, for example, from an antigen or an anti-idiotype antibody. The epitope region can also be derived from sugar chains like those which, for example, occur in glycated proteins. It can also be derived from lipid structures such as those which are present, for example, in phospholipids or lipoproteins. Therefore, the polyophene or binding partner in a polymeric form is made up of many identical or similar epitope regions and therefore has many similar binding sites for the sample antibody as already stated above. A binding site in the case of a protein as an antigen is understood as a peptide, the sequence of which is a part of the protein sequence of a protein antigen (analyte) and to which an antibody binds specifically, which is directed against this protein. In the case of an antigen which contains a sugar structure, the binding site could be the region of sugar molecule to which the sample antibody specifically binds. In the case of lipid structures, the lipid molecules can be the binding site of the sample antibody. A binding site can also be composed of combinations of peptide regions with sugars and / or lipids. However, polyhaptenes based on peptides are preferably used as binding partners in a polymeric form. In the case of binding partners in a polymeric form according to the invention, a high epitope density is of gen- eral importance, as the proteins of our 3fVI seem to bind specifically with high affinity to the binding partners in a polymeric form. The other criterion for the individual peptide components of the binding partners according to the invention in a polymeric form corresponds to the desired binding requirements in a monomeric form. Particles, for example latex, polystyrene, polyacrylate, polymethacrylate or gold, can be used as the carrier material for the polyhaptens. Polymers such as dextran or polypeptides such as polylysine, bovine serum albumin, / 5-galactosidase, non-specific immunoglobulins or fragments thereof can also be used as a carrier material for the polyhaptyne. The only condition when selecting the carrier is that it should not show cross-reactivity with antibodies in the sample liquid. An additional condition is that it should be possible to couple the haptens to the carrier. The epitope regions or the haptens are coupled to the carrier material by methods known to one of ordinary skill in the art, as described for example in EP-A-0 650 053 and WO 96/03652. In addition, spacer regions can be inserted between the epitope region and the carrier material, which is also described in the patent application open to the non-examined public mentioned above. All spacer regions known to one skilled in the art can be used. One requirement is that they are immunologically inactive, that is, they do not cross-react with antibodies in the sample.

Rj. it can be attached directly to the solid phase or indirectly bound to the solid phase by means of a specific binding system. The direct binding of Rx to the solid phase is carried out by methods known to a person familiar with the art. If Rx is indirectly bound to the solid phase by means of a specific binding system, then Rx is a conjugate which is constituted by a binding partner according to the invention in a polymeric form and a binding partner of a binding system. specific union. In this case, a specific connection system is understood as two associates which can react specifically with each other. In this case, the binding capacity can be based on an immunological reaction or on another specific reaction. A combination of biotin and avidin and or biotin and streptavidin is preferably used as a specific binding system. Other preferred combinations are biotin and antibiotin, hapten and antihapten, Fe fragment of an antibody and antibody against this fragment of Fe or carbohydrate and lectin. One of the reaction partners of this specifically univable pair is then a part of the conjugate that forms the receptor R? . The other reaction partner of the specific binding system is then present 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 those familiar with the art. In this case, a covalent bond as well as an absorptive bond is suitable. The solid phases which are particularly suitable are test tubes or microtiter plates made of polystyrene or similar plastics, the internal 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 particularly suitable, and are particularly preferred. Porous laminated carrier materials such paper can also be used as carriers. The receptor R2 is constituted of a molecule which reacts specifically with the sample antibody and a label. The molecule that specifically reacts with the sample antibody may be, for example, an antibody, an antibody fragment, a protein, an antigen or a hapten which specifically binds to the sample antibody. The only condition for the molecule as a component of R2 is that it reacts specifically with the sample antibody to be detected. Preferably, this molecule is a binding partner according to the invention in a polymeric form which specifically binds to the sample antibody. The binding partners in a polymeric form which are contained in R2 are prepared by the same methods as the binding partners in a polymeric form for R ,. An additional component of the R2 receiver is the label. A substance directly detectable as a label is preferably used, for example, a chemiluminescent, fluorescent or radioactive substance or a metallic sol, latex or gold particle. Also preferred are enzymes or other biological molecules such as labels, such as haptens. Digoxigenin is a particularly preferred label among haptens. The processes for labeling are familiar to a person skilled in the art and need not 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, latex or gold particle or by measuring the substance converted by the enzyme. The label can also be detected indirectly. In this case, an additional receptor which in itself in turn is coupled to a signal generating group, binds specifically to the tag of R2 such as a digoxigenin similar to hapten. 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. An antibody or antibody fragment can be used, for example, 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 is preferably digoxigenin or another hapten and the detection is carried out via a peroxidase-coupled antibody which is directed against digoxigenin or against the hapten. In order to reduce the antibodies with the IgG of the same antigen specificity, they are added to binding partners in a monomeric form according to the invention to the test mixture. The term "monomer" means that the binding partners in a monomeric form according to the invention contain only one epitope region or only one binding site for the antibody whose interference is to be reduced, i.e., a structure which reacts immunologically specifically with the IgG antibody. The monomeric structure of these binding partners is important in order to ensure that only antigen-specific IgG antibodies whose interference is to be reduced, bind to the binding statements in a monomeric form and not the IgM antibodies that are to be detect. The epitope region can be derived - as described above for binding partners in a polymeric form -, for example from an antigen or an antibody to idiotype. According to the prerequisites for binding partners in a polymeric form, the epitope regions of the binding partners in a monomeric form can also be derived from sugar and / or lipid structures or structures combined with peptide, lipid and / or peptide components. of sugar. All structures that can be derived from an epitope region which have a binding site to which the antibody of the IgG class binds specifically, whose interference will be reduced, in the presence of Ig antibodies of the same specificity, can be used. The only prerequisite for the binding site, that is, for the binding partners used in monomeric form, is that the specific capacity for IgG binding is retained. This condition also applies to the case where sugar or lipid structures are present at the binding site. According to the invention, it is also possible to use binding partners in a monomeric form which flank or overlap the binding site to which the IgG antibody specifically binds whose interference is to be eliminated. Therefore, it is also possible to eliminate the interference by IgG antibodies whose binding site includes an epitope which is not exactly identical to the epitope which is recognized by the IgM antibodies to be detected. These IgG antibodies may have a greater or lesser degree of cross-reactivity with the IgM antibodies to be detected. The addition of binding partner in a monomeric form which corresponds to the epitopes of these antibodies which cross-react and therefore have a high affinity for these, also eliminates the interference by these IgG antibodies. A mixture of binding partners in a monomeric form which overlaps to a greater or lesser degree the epitopes of the IgM antibodies to be detected and is preferably used to eliminate interference by IgG antibodies. Peptides that are preferably used as binding partners in a monomeric form. In the case of a protein such as an analyte, we mean a binding site - as well as the definition for the binding partner in a polymeric form - as a peptide, the protein sequence of a protein antigen and to which specifically binds an antibody directed towards this protein, which, in the case of the present invention, it is an IgG antibody. In addition to these peptides, it should also be understood that a binding site includes peptides with amino acid sequences which have an essentially equivalent binding specificity and / or affinity for the IgG antibody to be detected, such as the peptides mentioned in the foregoing. . These peptides can preferably be derived from the aforementioned peptides by substitution, deletion, insertion of individual amino acid residues. The peptides according to the invention which correspond to specific binding sites are also understood to include peptide derivatives in which one or more amino acids have been derivatized (derivatives have been formed) 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 side groups of reactive amino acids, for example the free amino groups, free carboxyl groups and / or free hydroxyl groups, have been derivatized. Specific examples of derivatives of amino groups are sulfonamides or carboximides, thiourethane derivatives and ammonium salts, for example hydrochloride. The derivatives of the carboxyl group are salts, esters and amides. Examples of derivatives of the hydroxyl group are 0-acyl or O-alkyl derivatives. The peptides are preferably produced by chemical synthesis according to methods known to a person familiar with the art and need not be elucidated especially here. In addition, the term "peptide derivative" also encompasses such peptides in which one or more amino acids have been substituted by naturally occurring or naturally occurring amino acid homologs of the "standard" twenty amino acids. Examples of such homologs are 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine, ornithine, alanine and 4-aminobutyric acid. Peptide derivatives must have an essentially equivalent binding or specificity for IgG antibodies whose interference will be reduced as the peptides from which they are derived. The peptides according to the invention which correspond to a specific binding site are also referred to as mimetic peptide substances termed antimimetics in the following, which have specificity or / and binding affinity essentially equivalent for the IgG antibodies whose interference goes to reduce as the peptides mentioned above or peptide derivatives. Peptide mimetics are compounds which can replace 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. The methods for the production of peptide mimetics are described in Giannis and Kolter, "Ángew. 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 9 and 12 amino acids. In the case of peptide mimetics 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 containing the epitope to which the IgG antibody specifically binds whose interference is to be reduced. However, additional flanking peptide sequences may be present which no longer correspond to the specific epitope, at the N-terminus and / or at the C-terminus of the peptide. This measurement may not be necessary to improve the solubility of the peptide. The only prerequisite is that the peptide as a binding partner in a monomeric form is actually present as a monomer and that the ability to strongly bind IgG antibodies whose interference is to be reduced is retained. A prerequisite for the use of the binding partners in a monomeric form (in this case: peptides) is that the same epitope is present in monomeric form than the epitope which is present on the binding partners in a polymeric form or the polyhaptens in a polymeric form. This means that, in order to effectively eliminate the interference, the IgG antibodies which bind to the binding partners in a monomeric form must have the same immunological specificity as the IgM antibodies to be detected. In a test for the determination of IgM antibodies, the interference of IgG antibodies of the same antigen specificity should always be eliminated.

The binding partners in a monomeric form are preferably used in an excess of 10 to 10,000 times compared to the concentration of the epitopes on the binding partners in polymeric form. The binding partners in a monomeric form are preferably used in an excess of 10 times to 1,000 times and particularly preferably in a 100-fold excess. The concentration of the binding partners in a monomeric form is limited only to the extent that the binding partners in a monomeric form are no longer soluble over a certain concentration. The concentration of the epitopes in the binding partners in a polymeric form depends on the size of the carrier material and can be easily determined individually by a person familiar in the art by any test procedure and any detection parameter. A guide value for the concentration of the epitopes in binding partners in a polymeric form of 5 to 500 ng peptide per ml of carrier material has been shown to be suitable in the case of peptide epitopes (length: 6 to 20 amino acids). In addition to the binding partners in a monomeric form, additional interference eliminating agents such as, for example, antibodies against Fd described in the introduction can also be used, in order to further reduce the interference by IgG antibodies.

Homogeneous as well as homogeneous methods can be used as test procedures for the antigen-specific IgM test. In a homogeneous test procedure, the complex consisting of R1 (shows IgM and R2 does not bind to a solid phase.) Instead, the agglutination of several such complexes with one another leads to a turbidity which is a measure for the concentration of IgM The binding partners in a monomeric form which reduces the IgG interference do not inhibit the agglutination However, a heterogeneous method is preferably carried out A method according to the bridge test principle ( see EP-A-0 280 211) is carried out in a particularly preferable manner In one of the preferred embodiments a conjugate of a polyhapten according to the invention and a binding partner of a specific binding system, preferably biotin it is used as RL R2 is composed of a polyhapten according to the invention or binding partner in a polymeric form and a label, preferably digoxigenin. they are particularly preferred used in Rx and R2. In this preferred modality, receptors x and R2 are incubated in the presence, in this preferred case, of a solid phase coated with avidin or streptavidin simultaneously with the sample and binding partners in a monomeric form, preferably peptides. In this process, the binding partners in a polymeric form of Rx and R2 react specifically with the IgM antibodies to be determined while the IgG antibodies of the same specificity are masked by the peptides, ie, by the associated • binding in a monomeric form so that they can not bind to the binding partners in a polymeric form. Therefore, the entire avidin / streptavidin-Rj ^ -IgM-sample antibody-R2 complex binds 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 (in this case with an antibody directed against digoxigenin) which specifically recognizes the label of R2. The additional receptor is coupled to a signal generating group preferably with the enzyme peroxidase. After an optional additional washing step, the sample antibody is detected via a signal generating group. In this case, through the substrate converted by the enzyme. In this test procedure, the incubation of the sample with Rlf R, and the binding partners in a monomeric form and the additional receptor can also be carried out concurrently. This further simplifies the test procedure. The test procedure described above is also very suitable for an application to automated systems. It is also possible to detect several antigen-specific IgM antibodies such as HIV antibodies against various HIV antigens. In such a case, the additional receiver can also be used as a universal label since the additional receiver specifically recognizes the R2 tag. If several IgM antibodies with different antigen specificities are simultaneously detected, the polyhapten components of Rx and R2 as well as the binding partners in a monomeric form that is used to reduce the interference should have the appropriate specificities. All biological fluids known to a person familiar with the art can be used as samples. Preferably bodily fluids such as whole blood, blood serum, blood plasma, urine, saliva, etc. are used as sample. In addition to the sample, the solid phase and the receptors mentioned above, other additives may be present in the test mixture 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 this person in a simple manner. A subject matter of the invention is also a reagent for the determination of a specific antigen antibody of the immunoglobulin class which, in addition to the usual test additives for immunoassays, contains binding partners in a monomeric form, i.e. preferably peptides and an Rx receptor capable of binding to the antibody to be determined, which is capable of binding to the solid phase and whose essential component is a binding partner in a polymeric form that is specifically recognized by the antibody that is leaving to determinate. A further subject matter of the invention is a reagent for the determination of an antigen specific antibody of the immunoglobulin M class which, in addition to the usual test additives for immunoassays, contains binding partners in a monomeric form, i.e. preferably peptides and two receptors R1 and R2 capable of binding to the antibody to be determined, of which Rx is capable of binding to a solid phase and R, presents a label where an essential component of each of the receptors R? and R2 is a binding partner in a polymeric form that is specifically recognized by the antibody to be determined. A further subject matter of the invention is the use of binding partners in a polymeric form for the specific determination of IgM antibody antigen by one of the methods mentioned before according to the invention. A further subject matter of the present invention is the use of binding partners in a monomeric form, ie, preferably peptides to reduce interference by IgG antibodies and / or rheumatoid factors in the determination of antigen-specific IgM antibodies. The invention was elucidated by the following example.

Example Antigen-specific IgM test: IgM against HIV 2 Multimeric antigens labeled with biotin and labeled with digoxigenin (HIV 2) are incubated with sample antibodies and a solid phase coated with streptavidin (incubation at 25 ° C or 37 ° C, c.a. 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-digoxigenin-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 at 25 ° C or 37 ° C for ca. 30 to 120 min, in this example: 60 min. ° C). 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 c. 0.5% protein / protein-derived additives (here lactalbumin peptone and BSA among others). The sample antibodies in this case are mouse monoclonal antibodies (IgM and IgG) against an HIV2 epitope diluted to ca. 2-10 μg / ml in human serum negative against HIV. The competition is carried out with a peptide antigen of HIV 2 unlabeled free in an excess of 10 to 100 times compared to the concentration of the peptide epitopes in the polyhaptens.

Test signal in mA: The addition of monomeric peptides makes the test for HIV 2 antibody specific for IgM. Some samples provide false positive test results without the addition of the peptide. It is noted that in relation to this date, the best method known to 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 (10)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A method for the determination of antigen-specific antibody of immunoglobulin class M by incubating the sample with at least two different sectors R1 and R2 in where both receptors are capable of specifically binding to the antibody, Rx binds or can bind to a solid phase and R2 carries a label, wherein an essentially Rx component is a binding partner in a polymeric form which is specifically recognized by the antibody to be determined, and the interference by IgG molecules of the same specificity present in the sample is reduced, by adding binding partners in a monomeric form.
2. 2. The method according to claim 1, characterized in that an essential component of R? and R2 is a binding partner, in a polymeric form which is specifically recognized by the antibody to be determined and the interference is reduced by IgG molecules of the same specificity that are present in the sample by adding binding partners in a monomeric form.
3. The method according to claim 1 or 2, characterized in that biotin / avidin, biotin / streptavidin, biotin / antiobiotin, hapten / antihapten, Fe fragment of an antibody / antibody against this fragment of Fe or carbohydrate / lectin are used as the specific binding system to join Rx to the solid phase.
4. The method according to one of claims 1 to 3, characterized in that the receptor R2 is labeled with quinolinescent, fluorescent or radioactive substances, or with an enzyme or another biological molecule.
5. The method according to one of claims 1 to 4, characterized in that the sample is incubated simultaneously with Rx and R2, and the binding partners in monomeric form are used to reduce the interference.
6. The method according to one of claims 5, characterized in that the test mixture is incubated with an additional receptor which binds specifically to the label of the receptor R2 in which the additional receptor is a conjugate of a specific receptor for the label of R- and a label, and subsequently the label is determined.
7. The method according to one of claims 6, characterized in that binding partners are added in a monomeric form used to reduce the interference, in an excess of 10 to 10,000 times compared to the concentration of the epitopes on the associates. of binding in a polymeric form of RL and R2.
8. 8. The reagent for the determination of an antigen-specific antibody of the immunoglobulin M class by a method as claimed in one of the previous claims, characterized in that in addition to the common test additives for immunoassays, it contains binding partners in a monomeric form and an Rx receptor capable of binding to the antibody to be determined, which is capable of binding to a solid phase and whose essential component is a binding partner in a polymeric form.
9. 9. A reagent for the determination of an antigen-specific antibody of the immunoglobulin class M by a method as claimed in one of the previous claims, characterized in that > in addition to the common test additives for immunoassays, it contains binding partners in a monomeric form and two receptors R and R2 capable of binding to the antibody to be determined, whose essential component is, in each case, a binding partner in a polymeric form and wherein R, is capable of binding to a solid phase and R2 has a label.
10. 10. The use of binding partners in a monomeric form to reduce interference by IgG antibodies and / or rheumatoid factors in the determination of an antigen-specific IgM antibody.