Immunoprecipitation assay using non-blood-based antibodies

Abstract

The present invention is directed to the field of in vitro diagnostics and relates to reagents containing non-blood-based antibodies for determining an analyte in an immunoprecipitation assay and having a sensitivity comparable to conventional reagents containing polyclonal antibody sera.

Description

[0001] 202314152 1 DESCRIPTION Immunoprecipitation Assay with Non-Blood-Based Antibodies The present invention lies in the field of in vitro diagnostics and relates to reagents containing non-blood-based antibodies for the determination of an analyte in an immunoprecipitation assay and with a sensitivity comparable to conventional reagents consisting of polyclonal antibody sera. Immunoprecipitation assays belong to the group of homogeneous immunoassays and are based on the interaction between antibodies and the antigen to be detected. The resulting immune complexes lead to aggregates and high-molecular-weight precipitates that absorb incident light in solution or scatter incident light, or exhibit altered diffusion behavior in gels.The turbidity of a reaction mixture caused by immune aggregates is essentially proportional to the amount of antigen contained in the reaction mixture and can be quantitatively determined as an increase in absorbance (turbidimetric) or as an increase in scattered light intensity at a specified angle relative to the incident light (nephelometric). The optimal conditions for performing such immunoprecipitation assays, such as buffers, detergents, neutral salts, accelerators, sample dilution, etc., are described in the literature and vary depending on the antibodies used and the antigens to be detected. The advantages of immunoturbidimetric and immunonephelometric methods are their relative ease of automation and low manufacturing costs, which have led to their widespread use in in vitro diagnostics.Due to their optimal cross-linking properties, animal antisera are primarily used in immunoprecipitation assays, such as sera from rabbits, horses, or mice immunized with an antigen. Such antisera ultimately contain a polyclonal mixture of various natural, blood-based antibodies directed against different epitopes of the antigen. The disadvantages of using animal antisera are, on the one hand, that quality fluctuations in in vitro diagnostic products are unavoidable, since each immunized animal produces an individual immune response and only a limited amount of antiserum is available. On the other hand, many animals are required to obtain the antisera, and their husbandry, treatment, and slaughter must comply with strict animal welfare regulations and, not least, also raise ethical concerns.To avoid these disadvantages, antibody mixtures containing various recombinant or monoclonal antibodies directed against different epitopes of an antigen (so-called "multiclonal antibody mixtures") are increasingly being used. These mixtures attempt to mimic a polyclonal antiserum. Using hybridoma technology, monoclonal antibodies can be produced in vitro, which significantly reduces animal consumption compared to the production of polyclonal antisera. Using phage display technology, antibody candidates can be selected in vitro and subsequently produced in in vitro expression systems ("recombinant antibodies"), completely eliminating the use of animals. The advantages of such so-called multiclonal antibody mixtures are the complete reproducibility of their composition and the greatest possible avoidance of the use and consumption of animals.The use of reagents containing multiclonal antibody mixtures enables the sensitive and specific determination of a wide variety of analytes in various immunoassay formats, such as ELISA assays or flow cytometry. However, when used in immunoprecipitation methods, particularly in turbidimetric or nephelometric immunoprecipitation methods, the sensitivity achieved is often not comparable to that achieved using animal antisera. This has so far excluded the animal-friendly use of non-blood-based or non-animal, multiclonal antibody mixtures in immunoprecipitation methods from widespread application in in vitro diagnostics. The present invention is therefore based on the object of exploiting the advantage of the animal-friendly use of non-blood-based orTo make non-animal, multiclonal antibody mixtures usable for immunoprecipitation methods, or to improve a reagent containing a mixture of at least two different antibodies from the group of monoclonal, genetically engineered, and synthetic antibodies and with specificities for different epitopes of an analyte so that a sensitive and specific determination of the analyte is also achieved in immunoprecipitation methods. This object is achieved by additionally adding one or more immunoglobulins without specificity for the analyte to the reaction mixture when using a reagent containing a mixture of at least two different antibodies from the group of monoclonal, genetically engineered, and synthetic antibodies and with specificities for different epitopes of an analyte in an immunoprecipitation method.This results in a sensitive and specific determination of the analyte using non-blood-based, multiclonal antibody mixtures in immunoprecipitation methods. Even if animal serum or animal-derived components cannot be completely dispensed with in immunoprecipitation methods in all cases, the solution according to the invention does, however, make it possible to dispense with the immunization of animals and the keeping and slaughter of immunized animals, so that the overall animal consumption for the production of reagents for the method according to the invention is reduced to a minimum.A first aspect of the invention is therefore an immunoprecipitation method for determining an analyte in a sample, wherein the sample is mixed with a mixture of at least two different antibodies from the group consisting of monoclonal, genetically engineered, and synthetic antibodies, and having specificities for different epitopes of the analyte, to form a reaction mixture. One or more immunoglobulins without specificity for the analyte are further added to the reaction mixture. An "immunoprecipitation method for determining an analyte" refers to all immunoassay formats based on the qualitative, semi-quantitative, or quantitative detection of high-molecular-weight immunoprecipitates, wherein the formation of the immunoprecipitates is based on an antigen-antibody interaction.In a preferred embodiment of the immunoprecipitation method according to the invention, the immunoprecipitates are measured in a liquid phase to determine the analyte by measuring the turbidity of the liquid phase. Particularly preferably, the turbidity due to the immunoprecipitates is measured turbidimetrically, i.e. by measuring the increase in absorbance of a liquid reaction mixture, or nephelometrically, i.e. by measuring the increase in scattered light intensity at a specified angle compared to incident light. A preferred embodiment is therefore a nephelometric or turbidimetric immunoprecipitation method. 202314152 5 The term “analyte” primarily encompasses substances that have immunogenic epitopes, such as proteins, peptides, nucleic acids, lipids, lipopolysaccharides, etc., and that can be directly bound by a specific antibody.In this case, the analyte is an antigen that can be bound by an antibody specific for this antigen. Typical examples of analytes of interest in in vitro diagnostics are plasma or serum proteins, such as antibodies that are present in abnormally high or low concentrations in certain disease states, or components of viruses or bacteria that indicate the presence of an infection with a pathogen. The term “sample” includes body fluid samples, particularly from humans and animals, such as blood, plasma, serum, urine, saliva or cerebrospinal fluid, but also stool samples or tissue samples. If necessary, the samples must be pretreated in order to make the analyte accessible for the detection method or to remove interfering sample components. Such pretreatment of samples can, for example,involve the separation and / or lysis of cells or the centrifugation of samples. According to the invention, the sample is mixed with a mixture of two or more different antibodies with specificities for different epitopes of the analyte to form a reaction mixture. Preferably, the sample is mixed with a mixture of more than two different antibodies with specificities for different epitopes of the analyte to form a reaction mixture, particularly preferably with three, four, five, six or more such different antibodies. Mixtures of four to six different antibodies have proven useful for imitating polyclonal sera. 202314152 6 The analyte-specific antibodies are selected from the group of monoclonal, genetically engineered and synthetic antibodies; these can be obtained largely without the use of animals.A “monoclonal antibody” is an antibody produced by a cell line that originates from a single, isolated cell. Such cell lines are typically established using hybridoma techniques, in which immune cells from immunized animals, such as a mouse or rabbit, are fused with myeloma cells to generate antibody-producing hybridoma cells, and suitable clones are then isolated and separated. The hybridoma cell lines obtained in this way are then available for the long-term production of monoclonal antibodies. Larger quantities of antibodies can be obtained, for example, from the cell culture supernatant from fermenters or roller cultures. A “genetically engineered antibody” (also called “recombinant antibody”) is an antibody produced by a genetically modified cell line.Such cell lines are typically established by transforming or transfecting isolated animal, human, plant, fungal, or prokaryotic cells with a nucleic acid encoding the antibody. Phage display techniques are suitable for locating nucleic acid sequences encoding antigen-specific antibodies or fragments thereof. The encoding nucleic acid sequences are then cloned into suitable vectors / plasmids, which are then introduced into the host cell. The genetically modified cell lines thus obtained are then available for the sustained production of genetically engineered antibodies. Larger quantities of antibodies can be obtained, for example, from the cell culture supernatant from fermenters or roller cultures. 202314152 7 A “synthetic antibody” is an antibody produced using a cell-free method.If the amino acid sequence of a suitable antibody is known, it can be produced in vitro by chemical Merrifield synthesis of peptides followed by protein ligation of the peptides. Another method is cell-free gene expression, in which an antibody-encoding RNA is generated on the basis of the known amino acid sequence / DNA sequence of a suitable antibody, e.g. by in vitro transcription. This RNA is then incubated with an amino acid mixture in a lysate or extract (so-called "translation extracts") from, for example, E. coli, insect cells, wheat germ, or CHO cells. An antibody produced in this way can then be isolated from the mixture. An analyte-specific antibody from the group of monoclonal, genetically engineered, and synthetic antibodies is typically an immunoglobulin, for example an immunoglobulin (Ig) of the class or subclass IgA, IgD, IgE, IgG1, or IgG. 2a , IgG 2b, IgG3, IgG4, or IgM. It has at least one binding site (often called a paratope) for an epitope (often also called an antigenic determinant) on an antigen. Such an epitope is characterized, for example, by its spatial structure and / or by the presence of polar and / or apolar groups. The binding site of the antibody is complementary to the epitope. The antigen-antibody reaction functions according to the so-called "lock and key principle" and is generally highly specific, i.e., the antibodies are able to distinguish small deviations in the primary structure, charge, spatial configuration, and steric arrangement of the antigen. In particular, the so-called "complementarity determining regions" of the antibody contribute to the binding of the antibody to the antigen. The antibody can completely correspond to an antibody that was originally produced, for example, by immunizing a human or an animal, such asMouse, rat, guinea pig, rabbit, horse, donkey, camel, sheep, goat or chicken, as part of the natural immune response and is now reproduced by hybridoma cell techniques or cloning and expression of the coding nucleic acid sequence. Alternatively, modified antibodies can be produced by genetic engineering methods, which are formed by a combination of immunoglobulin-coding sequences from different species ("chimeric antibodies"); e.g., the variable region of a mouse antibody is linked to the constant region of a human or rabbit antibody. According to the invention, one or more immunoglobulins without specificity for the analyte are further added to the reaction mixture.It has been found that the addition of at least one immunoglobulin without specificity for the analyte, or the addition of several immunoglobulins without specificity for the analyte, promotes the formation of high-molecular-weight immunoprecipitates in the reaction mixture and thus improves the sensitivity of the method, particularly in samples with low analyte concentrations. The term "immunoglobulins without specificity for the analyte" refers to immunoglobulins (or synonymously "antibodies") that—in contrast to the analyte-specific antibodies described above—have no specificity for the analyte, i.e., they do not specifically bind the analyte.They are typically obtainable from human or animal sera from individuals who have not been immunized with the analyte and therefore do not produce analyte-specific antibodies, or from cell lines or cell-free expression systems that produce human, animal, or chimeric antibodies known not to specifically bind the analyte. In one embodiment of the immunoprecipitation method according to the invention, the one immunoglobulin or the several immunoglobulins without specificity for the analyte that is / are added to the reaction mixture are selected from the group of immunoglobulin classes or subclasses IgA, IgD, IgE, IgGl, IgG. 2a , IgG 2b, IgG3, IgG4, and IgM. If a mixture of immunoglobulins is added, these can be included in the mixture in any conceivable combination with regard to the immunoglobulin classes. An immunoglobulin without specificity for the analyte can be an immunoglobulin isolated from human or animal serum, or a monoclonal immunoglobulin (analogous to a monoclonal antibody as described above), or a genetically engineered immunoglobulin (analogous to a genetically engineered antibody as described above), or a synthetic immunoglobulin (analogous to a synthetic antibody as described above). In a further embodiment of the immunoprecipitation method according to the invention, exactly one immunoglobulin without specificity for the analyte is added to the reaction mixture, for example a human or animal immunoglobulin G (IgG), M (IgM), A (IgA), E (IgE), or D (IgD), particularly preferably from rabbit or mouse.Particularly preferably, a genetically engineered immunoglobulin is used in this case, which has the advantage that the entire immunoprecipitation process can be carried out without the use of blood-based antibodies. In a further embodiment of the immunoprecipitation process according to the invention, a mixture of several immunoglobulins without specificity for the analyte is added to the reaction mixture, for example, a mixture of immunoglobulins selected from the group consisting of IgA, IgD, IgE, IgGl, and IgG. 2a , IgG 2b, IgG3, IgG4, and IgM, in turn selected from human and animal immunoglobulins, particularly preferably from rabbit or mouse. Particularly preferably, partially, very particularly preferably exclusively, genetically engineered immunoglobulins are used in this case, which has the advantage that the entire 202314152 10 immunoprecipitation method can be carried out on a reduced scale or entirely without the use of blood-based antibodies. In another embodiment of the immunoprecipitation method according to the invention, the mixture of several immunoglobulins without specificity for the analyte, which is added to the reaction mixture, consists of a human or animal normal serum, or of a mixture of human and one or more animal normal sera, or of a mixture of different animal normal sera. Preferred animal normal sera originate from rabbit or mouse.In the case of the addition of a mixture of normal sera, these can be contained in the mixture in any conceivable combination with regard to the species of origin (in particular human, mouse, rabbit) and in different or equal proportions. A normal serum also includes stabilized sera that have been filtered or otherwise pretreated for this purpose. In yet another embodiment of the immunoprecipitation method according to the invention, the mixture of several immunoglobulins without specificity for the analyte, which is added to the reaction mixture, consists of a purified preparation of immunoglobulins of one or more immunoglobulin classes from human serum or from animal serum, or of a mixture of such purified preparations. Preferred purified preparations of immunoglobulins are, for example, total IgG preparations, preferably from rabbit or mouse serum.Various methods for obtaining immunoglobulins from plasma or serum are well known. For example, immunoglobulin G can be enriched using an alcohol precipitation method (Cohn, EJ et al., Preparation and Properties of Serum and Plasma Proteins. IV. A System for the Separation into Fractions of the Protein and Lipoprotein Components of Biological Tissues and Fluids. J. Am. Chem. Soc. 68, 459-475, 1946; and Cohn, EJ et al., A System for the Separation of the Components of Human Blood: Quantitative Procedures for the Separation of the Protein Components of Human Plasma. J. Am. Chem. Soc. 72, 465-474, 1950; and US Pat. No. 3,597,409). Another known method consists in the combination of precipitation steps with Rivanol and ammonium sulfate (Horejisi, J. and Smetana, R., The Isolation of Gamma Globulin from Blood-Serum by Rivanol. Acta Med. Scand. Vol. 155, 65-70, 1956).In a preferred embodiment of the immunoprecipitation method according to the invention, the mixture of at least two different antibodies with specificities for different epitopes of the analyte and the one or more immunoglobulins without specificity for the analyte are mixed with the sample simultaneously in the form of a single reagent. Such a reagent is described in more detail below. Nevertheless, the immunoprecipitation method according to the invention can in principle also be carried out by, in a first step a), mixing the sample with the mixture of at least two different antibodies from the group of monoclonal, genetically engineered, and synthetic antibodies and with specificities for different epitopes of the analyte to form a reaction mixture, and then, in a second step b), adding one or more immunoglobulins without specificity for the analyte to the reaction mixture.It is also possible to carry out the two steps a) and b) in reverse chronological order. A further object of the present invention is the use of one or more immunoglobulins without specificity for the analyte to enhance the immunoprecipitation reaction in an immunoprecipitation method for determining the analyte in a sample. Yet a further object of the invention is a reagent for use in an immunoprecipitation method for determining an analyte, wherein the reagent contains a mixture of at least two different antibodies from the group consisting of monoclonal, genetically engineered, and synthetic antibodies and having specificities for different epitopes of the analyte, and additionally one or more immunoglobulins without specificity for the analyte. The reagent can be a liquid solution or a resuspensible lyophilizate of such a solution.The analyte-specific antibodies contained in the reagent are selected from the group of monoclonal, genetically engineered, and synthetic antibodies, as already explained above. A preferred reagent contains a mixture of two, three, four, five, six, or more different antibodies with specificities for different epitopes of the analyte. The one or more immunoglobulins contained in the reagent without specificity for the analyte (as explained in more detail above) are preferably selected from the group of immunoglobulin classes or subclasses IgA, IgD, IgE, IgGl, IgG. 2a , IgG 2b, IgG3, IgG4, and IgM. The one or more immunoglobulins without specificity for the analyte contained in the reagent can be monoclonal, genetically engineered, or synthetic immunoglobulins isolated from human or animal serum. The one or more immunoglobulins without specificity for the analyte contained in the reagent can originate from humans, mice, or rabbits. In one embodiment of the reagent, it contains exactly one immunoglobulin without specificity for the analyte. 202314152 13 In another embodiment of the reagent, it contains a mixture of several immunoglobulins without specificity for the analyte.The mixture of several immunoglobulins without specificity for the analyte can consist of a human or animal normal serum or a mixture thereof (as also described above), or it can consist of a purified preparation of immunoglobulins of one or more immunoglobulin classes from human serum or from animal serum, or of a mixture of such purified preparations. A particularly preferred reagent additionally contains a polar solvent, preferably γ-butyrolactam (2-pyrrolidone), because it is then particularly stable in the liquid state. A liquid reagent preferably contains up to 10 volume percent of the polar solvent. A further object of the present invention is the use of a reagent according to the invention for carrying out an immunoprecipitation method for determining an analyte in a sample.The use of a reagent according to the invention enables a simplified implementation of an immunoprecipitation method for determining an analyte in a sample by enabling the simultaneous addition of a mixture of at least two different antibodies with specificities for different epitopes of the analyte and one or more immunoglobulins without specificity for the analyte. The following examples and figures serve to illustrate the present invention and are not to be understood as a limitation. Regardless of the grammatical gender of a particular term, this includes persons of male, female, or other gender identity. 202314152 14 EXAMPLES EXAMPLE 1: Preparation of an anti-human IgG reagent according to the invention containing a mixture of various monoclonal anti-human IgG antibodies and animal serum orpurified animal immunoglobulins and use in an immunoprecipitation method for the determination of human IgG in serum samples Example 1a): Preparation of the reagents 1 mL of a mixture of four different monoclonal antibodies (produced by four different mouse hybridoma cell lines) (“MAB mix”), which specifically bind four different epitopes on the Fc part of the heavy chain of human immunoglobulin G (IgG), in PBS buffer, pH 7.4 (total protein concentration 2 mg / mL) was mixed with 1 mL of i) a crude normal rabbit serum pre-filtered through a 0.22 µm Millex-GV PVDF filter (Merck Millipore Ltd.); or ii) a crude normal rabbit serum pre-filtered through a 0.22 µm Millex-GV PVDF filter (Merck Millipore Ltd.) pre-filtered crude normal mouse serum; iii) PBS buffer, pH 7.4 (i.e., no addition of non-specific immunoglobulins); or iv) a BSA solution (40 mg / mL bovine serum albumin in PBS buffer, pH 7.4) (i.e., no addition of non-specific immunoglobulins); or v) a crude normal bovine serum pre-filtered through a 0.22 µm Millex-GV PVDF filter (Merck Millipore Ltd.); vi) a total immunoglobulin preparation from rabbit serum; vii) an immunoglobulin G preparation from rabbit serum; or 202314152 15 viii) a mixture of recombinant immunoglobulins G mixed with rabbit Fc backbone and stabilized by the addition of 2-pyrrolidone (final concentration 1.5% by volume). The immunoglobulin G preparation from rabbit serum was obtained by ion exchange chromatography with DEAE Sepharose from a total immunoglobulin preparation as described by Fishman, JB and Berg, EADescribed in 2019 (Purification of Antibodies: Diethylaminoethyl (DEAE) Chromatography, Cold Spring Harbor Protocol; doi:10.1101 / pdb.prot099135). Example 1b): Nephelometric immunoprecipitation assay for the determination of human IgG in serum samples. Serum samples from six healthy human donors (normal sera, NS 1-6) with human IgG concentrations within the reference range (7-16 g / L) and two deficient serum samples (MS 1 and 2) (each containing 0 g / L human IgG) were used as sample material, both without and with the addition of different amounts of human IgG by adding Privigen infusion solution (100 mg / mL normal human immunoglobulin, CSL Behring, Marburg). The assays were performed automatically as endpoint tests on the BN II system (Siemens Healthineers). For this purpose, the samples are first diluted 1:400 with a diluent solution (N-Diluent, Siemens Healthineers).100 µL of the diluted sample are then first mixed with 160 µL of a reaction buffer (N reaction buffer, Siemens Healthineers) and then with 40 µL of an anti-human IgG reagent (see reagents i) to v) in Example 1a)) in a reaction cuvette. The resulting light scattering is measured over a period of 360 seconds at +37 °C. The measured raw signal values ​​(signal [bit]) represent the measurement results. 202314152 16 The results were compared with the results of the commercially available N antiserum assay (Siemens Healthineers, Siemens material number (SMN) 10446296) for the determination of human IgG, which was also performed automatically on the BN II system. The N-antiserum assay for the determination of human IgG uses a liquid polyclonal rabbit serum (derived from rabbits immunized with highly purified human IgG) as the detection reagent. The results are presented in Table 1 and Table 2.Table 1 clearly shows that the MAB mix with the addition of normal mouse or rabbit serum demonstrates four times higher sensitivity for the detection of human IgG than the MAB mix without the addition or with the addition of normal bovine serum. The MAB mix with the addition of normal mouse or rabbit serum demonstrates a sensitivity comparable to that of the commercially available N antiserum assay. Without the addition of normal mouse or rabbit serum according to the invention, sufficient sensitivity is not achieved with the MAB mix, at least in the lower human IgG concentration range.

[0002] 202314152 17 Table 1: Comparison of test results using different anti-Human IgG MAK Mix reagents containing animal sera MAK-MIX Sample – N Anti- without with with with with Human serum Additive BSA Bovine Rabbit Mouse IgG against Normal -Normal- Normal [mg / mL] Human IgG serum serum -serum Signal MW [Bit] NS 1 2421 989 966 979 2809 2608 NS 2 949 47 45 40 921 839 NS 3 2611 1281 1322 1304 3182 2955 NS 4 3111 2146 2099 2216 4313 3981 NS 5 2327 962 951 948 2693 2501 NS 6 2538 1241 1191 1199 3059 2827 MS 1 1 4 6 9 7 5 MS 1 - 29 13 5 11 23 19 0.002 MS 1 - 138 24 12 5 125 114 0.004 MS 1 - 318 5 3 10 299 265 0.007 MS 1 - 889 44 39 31 847 786 0.014 MS 1 - 1678 267 265 233 1601 1481 0.029 MS 1 - 2875 2120 2281 2184 3452 3209 0.058 MS 1 – 4012 5760 6038 5819 7241 6724 0.116 MS 2 4 2 7 -2 6 5 MS 2 - 23 2 2 9 29 26 0.002 MS 2 - 129 19 5 11 131 109 0.004 MS 2 - 324 11 7 5 305 299 0.007 MS 2 - 899 27 25 24 826 757 0.014 MS 2 - 1601 249 251 253 1653 1531 0,029 MS 2 - 2801 2201 2093 2007 3423 3170 0.058 MS 2 - 3897 5978 5715 5991 7071 6289 202314152 18 Table 2 clearly shows that the MAB mix with the addition of purified rabbit immunoglobulins (total immunoglobulin and immunoglobulin G preparations) or a mixture of recombinant immunoglobulins G with rabbit Fc backbone shows a seven-fold higher sensitivity for the detection of human IgG than the MAB mix without additives. The MAB mix with the addition of the various rabbit immunoglobulins shows a sensitivity,which is comparable to the sensitivity of the commercially available N antiserum assay. Without the addition of rabbit immunoglobulins according to the invention, sufficient sensitivity is not achieved with the MAB mix, at least in the lower human IgG concentration range. Table 2: Comparison of test results when using different anti-human IgG MAB mix reagents containing rabbit immunoglobulins MAB MIX Sample – N Anti- without with with with rec. Human serum Additive Total Immune Immune IgG against Immunoglobulin globulins [mg / mL] Human globulin G from G with IgG from Rabbit Rabbit Rabbit Fc- Backbone Signal MW [Bit] NS 1 2421 989 2481 2466 2576 NS 2 949 49 901 933 966 NS 3 2611 1281 2666 2747 2723 NS 4 3111 2146 3626 3701 3769 NS 5 2327 962 2455 2528 2554 NS 6 2538 1241 2810 2921 3001 MS 1 1 4 5 7 4 MS 1 - 29 13 50 54 61 0.002 MS 1 - 138 4 181 190 202 0.004 MS 1 - 318 5 346 389 368 0.007 MS 1 - 889 44 871 903 922 0,014 202314152 19 MAK-MIX sample – N Anti- without with with with rec. Human serum Additive Total Immune Immune IgG against Immunoglobulin globulins [mg / mL] Human globulin G from G with IgG from Rabbit Rabbit Rabbit Fc- Backbone Signal MW [Bit] MS 1 - 1678 267 1655 1834 1902 0.029 MS 1 - 2875 2120 3507 3522 3589 0.058 MS 1 – 4012 5760 7402 7389 7289 0.116 MS 2 4 2 4 6 6 MS 2 - 23 2 53 50 54 0.002 MS 2 - 129 1 192 185 188 0.004 MS 2 - 324 11 375 378 381 0.007 MS 2 - 899 27 853 863 891 0.014 MS 2 - 1601 249 1709 1787 1866 0.029 MS 2 - 2801 2201 3522 3493 3541 0.058 MS 2 - 3897 5978 7266 7215 7173 0.116,

[0003] 202314152 20 EXAMPLE 2: Preparation of an anti-human IgG reagent according to the invention containing a mixture of various recombinant anti-human IgG antibodies and purified mouse and human immunoglobulins and use in an immunoprecipitation method for determining human IgG in serum samples Example 2a): Preparation of the reagents 1 mL of a mixture of four recombinant, genetically engineered antibodies (“Rek. MAK Mix”), which specifically bind four different epitopes on the Fc part of the heavy chain of human immunoglobulin G (IgG), in PBS buffer, pH 7.4 (total protein concentration 5 mg / mL) was mixed with 1 mL of i) an immunoglobulin G preparation from mouse serum (0.5 mg / mL); or ii) an immunoglobulin preparation from human serum (Privigen infusion solution, 0.05 mg / mL normal human immunoglobulin, CSL Behring, Marburg).The antigen-binding domains of the four recombinant anti-human IgG Fc antibodies were isolated using phage display technology from a recombinant phage library containing the variable parts of immunoglobulin light and heavy chains and were subsequently cloned into a rabbit IgG backbone. Each construct was then expressed in a HEK cell expression system. Example 2b): Nephelometric immunoprecipitation assay for the determination of human IgG in serum samples The nephelometric determination of human IgG was carried out as described in Example 1b). 202314152 21 The results are shown in Table 3. Table 3 clearly shows that the Rek. MAB mix with the addition of mouse IgG or even with a low dose of human Ig shows twice the sensitivity for the detection of human IgG as the Rek. MAK mix without additives. Table 3: Comparison of test results using different anti-human IgG recons.MAK mix reagents containing immunoglobulins from mouse or human R. ek. MAK-MIX Sample – without human IgG with mouse IgG with human Ig supplement [mg / mL] Signal MW [Bit] NS 1 989 2481 2466 N S 2 49 901 933N S 3 1281 2666 2747N S 4 2146 3626 3701N S 5 962 2455 2528NS 6 1241 2810 2921 M S 1 4 5 7 MS 1 - 0.002 13 50 54 M S 1 - 0,004 4 181 190 M S 1 - 0,007 5 346 389 MS 1 - 0.014 44 871 903 M S 1 - 0,029 267 1655 1834MS 1 - 0.058 2120 3507 3522 M S 1 – 0,116 5760 7402 7389MS 2 2 4 6 M S 2 - 0,002 2 53 50 M S 2 - 0,004 1 192 185 MS 2 - 0.007 11 375 378 M S 2 - 0,014 27 853 863MS 2 - 0.029 249 1709 1787 M S 2 - 0,058 2201 3522 3493M S 2 - 0,116 5978 7266 7215

Claims

202314152 22 CLAIMS 1. A reagent for use in an immunoprecipitation method for determining an analyte, the reagent comprising a mixture of at least two different antibodies from the group of monoclonal, genetically engineered and synthetic antibodies and having specificities for different epitopes of the analyte, characterized in that the reagent additionally contains one or more immunoglobulins without specificity for the analyte.

2. A reagent according to claim 1, comprising a mixture of three, four, five, six or more different antibodies with specificities for different epitopes of the analyte.

3. A reagent according to any one of the preceding claims, wherein the one or more immunoglobulins without specificity for the analyte are selected from the group of immunoglobulin classes or subclasses immunoglobulin G (IgG), G1 (IgG1), G2a (IgG 2a ), G2b (IgG 2b), G3 (IgG3), G4 (IgG4), M (IgM), A (IgA), E (IgE) and D (IgD).

4. Reagent according to one of the preceding claims, wherein the one or more immunoglobulins without specificity for the analyte are monoclonal, genetically engineered or synthetic immunoglobulins isolated from human or animal serum.

5. Reagent according to one of the preceding claims, which contains exactly one immunoglobulin without specificity for the analyte.

6. Reagent according to one of claims 1 to 4, which contains a mixture of several immunoglobulins without specificity for the analyte. 202314152 23 7. The reagent according to claim 6, wherein the mixture of several immunoglobulins without specificity for the analyte consists of a human or animal normal serum, or of a mixture of human and animal normal serum, or of a mixture of different animal normal sera.

8. The reagent according to claim 6, wherein the mixture of several immunoglobulins without specificity for the analyte consists of a purified preparation of immunoglobulins of one or more immunoglobulin classes from human serum or from animal serum, or of a mixture of such purified preparations.

9. The reagent according to any one of the preceding claims, wherein the one or more immunoglobulins without specificity for the analyte originate from humans, mice, or rabbits.

10. The reagent according to any one of the preceding claims, further comprising a polar solvent, preferably γ-butyrolactam. 11.Use of a reagent according to any one of claims 1 to 10 for conducting an immunoprecipitation method for determining an analyte in a sample.

12. An immunoprecipitation method for determining an analyte in a sample, wherein the sample is mixed with a mixture of at least two different antibodies from the group consisting of monoclonal, genetically engineered, and synthetic antibodies and having specificities for different epitopes of the analyte to form a reaction mixture, characterized in that one or more immunoglobulins without specificity for the analyte are further added to the reaction mixture. 202314152 24 13. The immunoprecipitation method according to claim 12, wherein the mixture of at least two different antibodies with specificities for different epitopes of the analyte and the one or more immunoglobulins without specificity for the analyte are mixed with the sample simultaneously, in the form of a single reagent.

14. The immunoprecipitation method according to claim 13, wherein a reagent according to any one of claims 1 to 10 is mixed with the sample.

15. The immunoprecipitation method according to any one of claims 12 to 14, wherein, for the determination of the analyte in the sample, the immunoprecipitates formed in the reaction mixture are measured turbidimetrically or nephelometrically.