Method and kit for reducing interferences in immunoassays

Abstract

To provide a method and a kit for reducing interferences in immunoassays.SOLUTION: The present invention relates to the field of immunoassays for in vitro diagnostic applications and the use of a test-specific blocking antibody for reduction of interferences caused by heterophilic antibodies or rheumatoid factors.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] The present invention relates to the field of immunoassays for in vitro diagnostic applications and to the use of assay-specific blocking antibodies directed to reducing interference caused by heterophilic antibodies or rheumatoid factors.

Background Art

[0002] Immunoassays have been used for decades in clinical diagnostic tests to quantitatively or qualitatively detect various analytes in body fluid samples. Antibodies used for direct detection of antigens or indirect detection of different analytes are usually monoclonal or polyclonal animal antibodies obtained from immunized animals (e.g., rabbits, mice, sheep) or by biotechnological means. False test results caused by interfering substances derived from patient samples can lead to a wide range of misdiagnoses. One well-known and relatively frequent problem is the problem of interfering antibodies that may be intrinsically present in an individual's sample (Non-Patent Document 1). Examples of interfering antibodies are heterophilic antibodies, i.e., antibodies in a patient's blood directed against antigens of another species, particularly immunoglobulins, such as human anti-mouse antibodies (HAMA). Another example of an interfering antibody is the so-called rheumatoid factor, i.e., a human autoantibody directed against the Fc portion of human immunoglobulin G. All of these interfering antibodies usually have an affinity for animal antibodies and often bind to the Fc portion. If an animal antibody ("assay antibody") is used as part of a detection reaction for the detection of an analyte in an assay system, what can occur in the presence of interfering antibodies from a patient sample is a binding reaction between the assay antibody and the interfering antibody, and this binding reaction can block the detection reaction and result in false negative / wrongly low results, or enhance the detection reaction and result in false positive / wrongly high results.

[0003] Therefore, in order to reduce such interference, it is a standard procedure to add blocking antibodies to the latest immunoassays. The blocking antibodies are usually a mixture of randomly selected non-specific antibodies of the same immunoglobulin class and the same species as the analytical antibodies used in the test system. For example, in a test system that uses a monoclonal mouse antibody (e.g., mouse IgG1) as the analytical antibody, it can be further added with irrelevant mouse IgG1, that is, an excessive mixture of mouse IgG1 that is non-functional in the test system. In most cases, this causes any interfering antibodies from the patient sample to bind to the irrelevant antibodies, thereby preventing or at least minimizing the problematic binding to the analytical antibodies. Such blocking agents are commercially available, for example, the Heterophilic Blocking Reagent (HBR) reagent manufactured by Scantibodies Laboratory, Inc. or the TRU Block reagent manufactured by Meridian Bioscience, Inc. The blocking effect of such irrelevant antibodies can be optimized by pre-aggregating the irrelevant antibodies (Patent Document 1).

[0004] Despite these measures, there are still cases where a specific sample cannot be accurately analyzed using a specific immunoassay test system due to clearly insufficient blocking by conventional blocking reagents. In Non-Patent Document 2, the use of two different test systems for determining von Willebrand factor (VWF) activity, both of which involve the use of monoclonal mouse antibodies in particular, describes patient samples in which false high results were repeatedly obtained despite the addition of a HAMA blocking agent (for blocking human anti-mouse antibodies).

[0005] As used in connection with the present invention, an "immunoassay" is a method for detecting an analyte in a sample that involves the use of at least one antigen-specific antibody. The antigen-specific antibody can be, but need not necessarily be, a specific antibody for the analyte.

[0006] Depending on the assay setup, the antibodies used can perform various functions. For example, an antibody can be used as a capture antibody or as a labeled secondary antibody for directly binding and detecting the analyte; for this purpose, the antibody needs to be a specific antibody for the analyte. In another case, an antibody can be used, for example, to immobilize the binding partner of the analyte to be detected on a solid phase; for this purpose, the antibody needs to be an antibody specific for the said binding partner. Various immunoassay principles are known (direct method, indirect method, competitive method, non-competitive method). What is common to all is that such principles involve the use of at least one antigen-specific antibody that is directly or indirectly involved in an analyte-specific detection reaction in a selected assay system for detecting the analyte.

[0007] Thus, a conventional immunoassay optimized to minimize the occurrence of antibody-induced interference is a method for detecting an analyte in a body fluid sample that essentially comprises the following steps: i) preparing a reaction mixture by contacting the sample with a) an antigen-specific antibody that specifically binds to the antigen, and b) a mixture of non-specific, randomly selected antibodies that are empirically known to block the binding of interfering antibodies present in the sample to the antigen-specific antibody ; ii) measuring a measurement variable in the reaction mixture that is affected by the formation of a complex between the antigen and the first antigen-specific antibody and correlates with the amount of analyte.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Non-Patent Document

[0009]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0010] Despite the addition of a blocking antibody to the reaction mixture, there are always samples that cannot be accurately analyzed in a specific immunoassay due to apparently insufficient blocking. Therefore, an object of the present invention is to provide further methods and means for immunoassays that improve the reliability of immunoassays by effectively reducing any interference caused by heterophilic antibodies or interfering antibodies such as rheumatoid factors present in patient samples.

Means for Solving the Problems

[0011] This object is achieved by the present invention by using a blocking antibody derived from an analytical antibody and having a structure substantially identical to that of the analytical antibody. As a result, any interfering antibodies derived from patient samples that clearly bind highly specifically to the epitope of the analytical antibody and are thus not blocked or not sufficiently blocked by a randomly selected classical blocking antibody mixture are specifically captured in this case by binding to the induced blocking antibody and are consequently blocked. Antibodies having an amino acid sequence identical to that of the analytical antibody except for 1 to 3 modified amino acid residues and thereby having a significantly reduced antigen-binding ability compared to the analytical antibody result in efficient blocking of interfering antibodies and thus have been found to significantly improve the reliability of immunoassays.

[0012] Accordingly, the present invention provides a kit for use in a method of detecting an analyte in a body fluid sample. The kit comprises i) a first antigen-specific antibody, having a first amino acid sequence, specifically binding to an antigen, and the use of which results in an analyte-specific detection reaction in a defined assay system for the detection of an analyte, the first antigen-specific antibody (the "analytical antibody"), and ii) an antibody variant, having a second amino acid sequence, the antigen-binding ability of the antibody variant being significantly reduced compared to the first antibody or its competition with the first antigen-specific antibody for binding to the antigen being very low, such that the additional use of the antibody variant in a defined assay system for the detection of an analyte results in a maximum 15% reduction in the analyte-specific detection reaction, the antibody variant (also referred to as the "non-analytical antibody" or "non-analytical antibody variant") and containing the amino acid sequence of the antibody variant is identical to that of the first antigen-specific antibody except for 1 to 3 modified amino acid residues.

Brief Description of the Drawings

[0013]

Figure 1A

Figure 1B

Figure 2

Mode for Carrying Out the Invention

[0014] A kit for use in a method of detecting an analyte in a body fluid sample typically contains one or more reagents in liquid or lyophilized form or in the form of a coated solid phase, which are brought into contact with the body fluid sample to be analyzed (e.g., whole blood, plasma, serum, urine) so as to cause a detection reaction to enable a quantitative, semi-quantitative or qualitative determination of the amount or activity of the analyte.

[0015] The first antigen-specific antibody specifically binds to the antigen and is essential for generating the analyte-specific detection reaction targeted by the assay system (the "assay antibody"). The antigen-specific antibody can be a specific antibody of the analyte that specifically binds to the analyte derived from the body fluid sample. Alternatively, the antigen-specific antibody may specifically bind to a binding partner of the analyte. In this case, the binding partner of the analyte may be endogenous to the body fluid sample or may be added to the reaction mixture. In another embodiment of the detection method, the antigen-specific antibody may specifically bind to a cleavage product of the analyte.

[0016] Depending on the assay settings, the antibodies used can perform various functions. For example, the antibody can be used as a capture antibody or as a labeled secondary antibody for directly binding and detecting the analyte; for this purpose, the antibody needs to be a specific antibody of the analyte. In another case, the antibody can be used, for example, to immobilize the binding partner of the analyte to be detected on a solid phase; for this purpose, the antibody needs to be an antibody specific for the said binding partner. The principles of various immunoassays are known (direct method, indirect method, competitive method, non-competitive method). What is common to all is that such principles involve the use of at least one antigen-specific antibody that is directly or indirectly involved in the analyte-specific detection reaction in the selected assay system for detecting the analyte.

[0017] The first antigen-specific antibody can belong to any immunoglobulin class (IgA, IgD, IgE, IgG or IgM); its origin can be human, mouse, rabbit, mouse, sheep, camel or other animals. Preferably, the antibody is a monoclonal antibody or a recombinantly produced antibody. The first antigen-specific antibody may also be a chimeric antibody or a humanized antibody. The term "first antigen-specific antibody" explicitly includes not only whole antibodies but also various antigen-binding antibody fragments, such as Fab fragments or F(ab)2 fragments.

[0018] In various embodiments, the first antigen - specific antibody may be associated with a solid phase and / or components of a signal - forming system.

[0019] The term "solid phase" as used in the present invention refers to an article made of a porous and / or non - porous water - insoluble material, which can take various forms, such as containers, small tubes, microtiter plates (ELISA plates), beads, microparticles, rods, strips, filter paper or chromatography paper. Generally, the surface of the solid phase is hydrophilic or can be made hydrophilic. The solid phase can be composed of various materials, such as inorganic and / or organic materials, synthetic materials, natural materials and modified natural materials. Examples of solid - phase materials are polymers, such as cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, cross - linked dextran molecules, agarose, polystyrene, polyethylene, polypropylene, polymethacrylate or nylon; latex; ceramics; glass; metals, especially noble metals such as gold and silver; magnetite; mixtures or combinations thereof. Particles, including magnetic particles and latex particles, can be labeled with dyes, sensitizers, fluorescent substances, chemiluminescent substances, isotopes or other detectable labels.

[0020] "Components of a signal - forming system" are molecules that can generate a signal by themselves or can induce the generation of a signal, such as fluorescent substances, chemiluminescent substances, radioactive substances, or enzymes. The signal can be detected or measured, for example, based on enzyme activity, luminescence, light absorption, light scattering, emitted electromagnetic radiation or radioactive radiation or chemical reactions.

[0021] Suitable components of the signal formation system are, for example, the following: as enzymes, horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, glucose oxidase, β-galactosidase, luciferase, urease and acetylcholinesterase; enzyme substrates; dyes; as fluorescent substances, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, ethidium bromide, 5-dimethylaminonaphthalene-1-sulfonyl chloride and fluorescent chelates of rare earth elements; as chemiluminescent substances, luminol, isoluminol, acridinium compounds, olefins, enol ethers, enamines, aryl vinyl ethers, dioxenes, aryl imidazoles, lucigenin, luciferin and equorin; as sensitizers, eosin, 9,10-dibromoanthracene, methylene blue, porphyrins, phthalocyanins, chlorophyll, rose bengal; coenzymes; as radioisotopes, 125 I, 131 I, 14 C, 3 H, 32 P, 33 P, 35 S, 51 Cr, 59 Fe, 57 Co and 75 Se.

[0022] The term "associated" should be understood broadly and includes, for example, covalent and non-covalent bonds, direct and indirect bonds, adsorption to a surface, and encapsulation in a cavity. In the case of a covalent bond, the first antigen-specific antibody or antigen-specific antibody fragment is bound to the solid phase or to a component of the signal formation system via a chemical bond. An example of a non-covalent bond is surface adsorption. In addition to direct binding, the first antigen-specific antibody or antigen-specific antibody fragment can also be indirectly bound to the solid phase via a specific interaction with another binding partner, for example, via a specific interaction with avidin if the first antigen-specific antibody or antigen-specific antibody fragment is biotinylated.

[0023] The antibody variant of the kit according to the present invention is an antibody (also referred to as a "non-analytical antibody" or a "non-analytical antibody variant") derived from a first antigen-specific antibody or a corresponding antibody fragment, and 1.) an amino acid sequence that is identical to the amino acid sequence of the first antigen-specific antibody except for 1 to 3 modified amino acid residues, and 2.) an antigen-binding ability that is significantly reduced compared to the first antigen-specific antibody, such that the addition of the antibody variant in a defined assay system for the detection of an analyte results in a maximum 15% reduction of the analyte-specific detection reaction.

[0024] Thus, such "non-analytical" antibody variants are custom-made for a first antigen-specific antibody ("analytical antibody") and define a modified amino acid sequence from the known amino acid sequence of the analytical antibody (or after the amino acid sequence of the analytical antibody has been determined) through substitution, deletion, insertion, or chemical derivatization of one to three amino acid residues, and accordingly, the modified antibody variant is recombinantly produced and is usually available. The position of one modified amino acid residue or the positions of two or three modified amino acid residues are related to antigen binding and will be functionally restricted or inactivated by the modification of the amino acid sequence, and should be selected to be in the region of the analytical antibody such that the antigen-binding ability of the produced modified non-analytical antibody variant is lacking or at least significantly reduced only. For this purpose, it is preferred that one or more of the complementarity-determining regions (CDRs) of the heavy or light chain of the analytical antibody are modified by one to three amino acid residues, i.e., substituted, deleted, inserted, or chemically derivatized. The complementarity-determining regions (CDRs) of the heavy chain of the antibody (CDR-H1, CDR-H2, and CDR-H3) and the complementarity-determining regions (CDRs) of the light chain of the antibody (CDR-L1, CDR-L2, and CDR-L3) (according to the Kabat numbering scheme), which are separated from each other by the so-called framework regions, are well known to those skilled in the art. Particularly preferably, at least one amino acid residue within the complementarity-determining region of the heavy chain of the analytical antibody is modified. More preferably, at least one amino acid residue within the complementarity-determining region CDR-H3 of the heavy chain of the analytical antibody is modified.

[0025] Thus, in one embodiment of the test kit, there is an antibody variant in which one to three modified amino acid residues are located in one or more of the complementarity-determining regions (CDRs) of the heavy or light chain of the antibody variant.

[0026] In a further embodiment of the test kit, there is an antibody variant in which at least one modified amino acid residue is located in one complementarity-determining region of the heavy chain of the antibody variant.

[0027] In yet another embodiment of the assay kit, there is an antibody variant in which at least one modified amino acid residue is located in the complementarity determining region CDR-H3 of the heavy chain of the antibody variant.

[0028] The "modified amino acid residue" should be understood to mean an amino acid residue that is substituted, deleted, inserted or chemically derivatized in relation to consecutive positions of the amino acids of the primary sequence of the analytical antibody. In the case of substitution, the original amino acid residue is replaced by a different amino acid residue. Preferably, the substitution is a non-conservative substitution, i.e., a substitution between different families of amino acids that differ in terms of their side chains and chemical properties. Examples of different families are amino acids with basic side chains, amino acids with acidic side chains, amino acids with non-polar aliphatic side chains, amino acids with non-polar aromatic side chains, amino acids with polar side chains, amino acids with uncharged polar side chains, amino acids with charged side chains, amino acids with small side chains, amino acids with large side chains, etc. For example, a small amino acid residue is replaced by a large amino acid residue, or a charged amino acid residue is replaced by an uncharged amino acid residue.

[0029] The "non-analytical" antibody variant is also a naturally occurring variant of the first antigen-specific antibody, in which case the amino acid sequence of the variant has 1 to 3 substituted, deleted or inserted amino acid residues compared to the original analytical antibody.

[0030] Thus, the antibody variant is a non-functional variant of it or at least a less functional variant of it with respect to antigen-binding ability compared to the first antigen-specific antibody.

[0031] The antigen-binding ability of the antibody variant needs to be significantly reduced compared to the first antigen-specific antibody, such that the addition of the antibody variant in a defined assay system for the detection of an analyte results in a maximum 15% reduction of the analyte-specific detection reaction.

[0032] Reduction of the antigen-binding ability of an antibody variant can be measured in a comparative experiment with a first antigen-specific antibody in a standard assay for determining the specificity of binding to a target antigen, such as an ELISA assay, a BIAcore assay, an Octet BLI assay, or, if the antigen is expressed on the cell surface, a FACS-based assay.

[0033] However, it is extremely important to test the antigen-binding ability of an antibody variant in the same defined assay system for detecting an analyte in which the first antigen-specific antibody is used as the "analytical" antibody. The term "defined assay system" refers to an assay setting defined with respect to the components and method steps used. By varying a single component or a single method step in an assay setting that is otherwise unchanged, it becomes possible to determine the effect of the variation in an assay system that is otherwise defined. Ideally, the method for detecting the analyte is used as a defined assay system, in which case the use of the first antigen-specific antibody and the antibody variant is intended for its implementation. In the context of the present invention, what is used for this purpose is the first antigen-specific antibody for detecting the analyte, and additionally, i.e., in combination with the first antigen-specific antibody, the antibody variant to be tested.

[0034] In an assay system where the first antigen-specific antibody is used as the "assay" antibody, a suitable antibody variant does not have a significant competing effect on the analyte-specific detection reaction. This is ensured by first measuring the reaction intensity of the analyte-specific detection reaction over the entire measurement range of the assay system both with and without the addition of the antibody variant to the reaction mixture in a sample free of interfering antibodies. A suitable antibody variant is an antibody variant having a significantly reduced antigen-binding ability as compared to the first antigen-specific antibody, such that as a result, its presence does not reduce the reaction intensity of the analyte-specific detection reaction by more than 15%, preferably by more than 10%, and particularly preferably by more than 5%; the reduction in the antigen-binding ability of the antibody variant is functionally demonstrated by the fact that there is little or no competition between the antibody variant and the first antigen-specific antibody for binding to the antigen, i.e., little or no competition for the binding site of the antigen, such that as a result, using the antibody variant in combination with the first antigen-specific antibody does not unduly problematically affect the analyte-specific detection reaction, but rather is functionally demonstrated by showing that it results in a reduction of up to 15% in the reaction intensity of the analyte-specific detection reaction.

[0035] In the kit according to the invention, the first antigen-specific antibody and the antibody variant may be present in different reagents or in a single reagent.

[0036] In one embodiment of the kit, the first antigen-specific antibody is associated with a solid phase, for example on the surface of a container (as described above), for example on the bottom of a well of a microtiter plate, or inside a reaction tube. Such a kit is particularly suitable for performing heterogeneous assay methods, such as ELISA assays. Such a kit preferably further contains a further container containing the antibody variant as a component of a liquid reagent (or its lyophilized product).

[0037] In another embodiment of the kit, the first antigen-specific antibody is associated with the surface of the particulate solid phase (as described above). For this purpose, the kit contains a container containing the corresponding reagent in the form of a liquid suspension or a resuspendable lyophilized product. Such test kits are suitable for measuring agglutination using photometry.

[0038] In yet another embodiment of the kit, the first antigen-specific antibody is associated with a component of the signal-forming system (as described above). The first antigen-specific antibody may be directly associated with a component of the signal-forming system or may be indirectly associated, for example, if the antibody and the component of the signal-forming system are associated with a single solid phase, such as latex particles. For this purpose, the kit contains a container containing the corresponding reagent in liquid form or as a resuspendable lyophilized product. Depending on the nature of the signal-forming system, such test kits are suitable for measuring, for example, chemiluminescence, fluorescence or changes in absorption.

[0039] In a particularly preferred kit, the first antigen-specific antibody is an antibody specific for the analyte. In this case, the antibody is used directly as a capture antibody or, for example, as a labeled secondary antibody for binding and detecting the analyte in a sandwich immunoassay.

[0040] In another embodiment of the kit, in addition to the first antigen-specific antibody, for example in a kit used in a sandwich immunoassay, a different second antigen-specific antibody is additionally present, in which case the second antigen-specific antibody may be specific for the same antigen as the first antigen-specific antibody or may be specific for a different antigen. In such a kit, preferably, there is a further "non-analytical" antibody variant (as described above) which is a non-functional variant of it or at least a less functional variant of it with respect to antigen-binding ability, compared to the second antigen-specific antibody. Thus, such a kit c) a second antigen-specific antibody, A second antigen-specific antibody having a third amino acid sequence, specifically binding to an antigen, and its use resulting in an analyte-specific detection reaction in a defined assay system for the detection of an analyte, and d) A further antibody variant, having a fourth amino acid sequence, the antigen-binding ability of the further antibody variant being significantly reduced as compared to the second antigen-specific antibody, so that the additional use of the further antibody variant in a defined assay system for the detection of an analyte results in a maximum 15% reduction of the analyte-specific detection reaction, and the further antibody variant is additionally contained, the amino acid sequence of the further antibody variant being identical to the amino acid sequence of the second antigen-specific antibody, except for 1 to 3 modified amino acid residues.

[0041] Preferably, in the kit according to the invention, each antigen-specific antibody present is provided together with an antibody variant which is a non-functional variant thereof or at least a less functional variant thereof with respect to antigen-binding ability as compared to the respective antigen-specific antibody (as described above).

[0042] The invention further provides for the use of the kit according to the invention in a method for detecting an analyte in a body fluid sample.

[0043] Particularly preferred is the use of the kit according to the invention for the interference-free detection of an analyte in a body fluid sample containing interfering antibodies, for example from the group of heterophilic antibodies and autoantibodies.

[0044] In a specific embodiment of the kit, the first antigen-specific antibody is an antibody that specifically binds to the glycoprotein Ib (GPIb) protein. The GPIb protein is a binding partner of von Willebrand factor (VWF) and is used in various assays for the determination of VWF activity (see, for example, WO2009 / 007051A2). Qualitative or functional defects in VWF are detected through a reduction in the binding of VWF present in the sample to the added GPIb protein. The ability of VWF to bind to the added GPIb protein can be quantitatively determined, for example, by constructing an assay method such that the formation of a complex between VWF and GPIb in the assay reaction can be measured by measuring the aggregation of latex particles that aggregate only when they are coated with an anti-GPIb antibody and the VWF-GPIb complex is formed in the assay reaction and then bound by the anti-GPIb antibody associated with the latex particles. As described above, in such assays using mouse monoclonal anti-GPIb antibodies, it has been observed that false high results can be obtained repeatedly despite the addition of an HAMA blocker (for blocking human anti-mouse antibodies).

[0045] In a preferred form of the specific embodiment of the kit, · The first antigen-specific antibody is an antibody that specifically binds to the glycoprotein Ib (GPIb) protein and has an amino acid sequence according to SEQ ID NO: 1 (DTMIKGHYVMDY) in the complementarity-determining region CDR-H3 (according to the Kabat numbering scheme) of the heavy chain, · The antibody variant is an antibody whose amino acid sequence is identical to the amino acid sequence of the first GPIb protein-specific antibody except for two modified amino acid residues and has an amino acid sequence according to SEQ ID NO: 2 (DTMIKGHSVFDY) in the complementarity-determining region CDR-H3 (according to the Kabat numbering scheme) of the heavy chain.

[0046] Such test kits are suitable for use in methods for detecting VWF activity in a body fluid sample and have the particular advantage of enabling the interference-free detection of VWF activity in a body fluid sample containing interfering antibodies, for example, from a group of heterophilic and autoantibodies.

[0047] The present invention further provides a method for detecting an analyte in a body fluid sample, the method comprising: a) preparing a reaction mixture by mixing the sample with i. a first antigen-specific antibody having a first amino acid sequence, specifically binding to an antigen, the use of which results in an analyte-specific detection reaction in a defined assay system for detecting the analyte, the first antigen-specific antibody, and ii. an antibody variant having a second amino acid sequence, the antigen-binding ability of the antibody variant being significantly reduced compared to the first antibody, such that the additional use of the antibody variant in a defined assay system for detecting the analyte results in a maximum 15% reduction in the analyte-specific detection reaction, the antibody variant ; b) measuring a measurement variable in the reaction mixture that is affected by the formation of a complex between the antigen and the first antigen-specific antibody and correlates with the amount of analyte ; wherein also (as already mentioned above), the amino acid sequence of the antibody variant is identical to the amino acid sequence of the first antigen-specific antibody, except for 1 to 3 modified amino acid residues.

[0048] Preferably, the sample is first mixed with the antibody variant and the mixture thus produced is incubated, and only then is the first antigen-specific antibody added to the mixture. Pre-incubating the sample with the "non-analytical" antibody variant in this way results in particularly efficient blocking of interfering antibodies because the interfering antibodies are already bound before contacting the "analytical" antibody.

[0049] In one embodiment of the method according to the invention, the first antigen-specific antibody is an analyte-specific antibody, and the measurement variable to be measured is affected by the formation of a complex between the analyte and the first analyte-specific antibody. An example thereof is an immunoassay, in which case a complex of the analyte and a latex particle-associated analyte-specific antibody is formed, and the formation of the complex is determined photometrically based on the aggregation reaction of the latex particles in the reaction mixture.

[0050] In another embodiment of the method according to the invention, the first antigen-specific antibody is an antibody having specificity for a binding partner of the analyte, and the measurement variable to be measured is affected by the formation of a complex between the analyte, the binding partner of the analyte, and the first antigen-specific antibody having specificity for the binding partner of the analyte. An example thereof is a functional binding assay, in which case what is measured is not the amount of the analyte but rather the ability of the analyte to bind to a specific binding partner. In this case, a complex of the analyte, the binding partner of the analyte, and, for example, a latex particle-associated antibody having specificity for the binding partner is formed, and the formation of the complex is determined photometrically based on the aggregation reaction of the latex particles in the reaction mixture.

[0051] A specific embodiment of the method according to the invention is a method for detecting the activity of von Willebrand factor in a body fluid sample, in which case the first antigen-specific antibody is an antibody having specificity for the GPIb protein, and the measurement variable to be measured is affected by the formation of a complex between von Willebrand factor, the GPIb protein, and the first antigen-specific antibody having specificity for the GPIb protein.

[0052] In the method according to the invention, the first antigen-specific antibody may be associated with a particulate solid phase, and the aggregation of the particulate solid phase in the reaction mixture, which is affected by the formation of a complex between the antigen and the first antigen-specific antibody and correlates with the amount of the analyte, can be measured.

[0053] Aggregation of the particulate solid phase in the reaction mixture can be measured by photometry, for example, by turbidimetry or nephelometry. Binding assays based on the principle of particle-enhanced light scattering have been known since around 1920 (for reviews, see Newman, D. J. et al., Particle enhanced light scattering immunoassay. Ann Clin Biochem 1992;29:22-42). In this regard, polystyrene particles having a diameter of 0.1 to 0.5 μm are preferred, and polystyrene particles having a diameter of 0.15 to 0.35 μm are more preferred. It is preferred to use polystyrene particles having a functional group of amine, carboxyl or aldehyde. It is also preferred to use core-shell type particles. For the synthesis of particles and the covalent bonding of ligands, see, for example, Peula, J. M. et al., Covalent coupling of antibodies to aldehyde groups on polymer carriers. Journal of Materials Science: Materials in Medicine 1995;6:779-785.

[0054] Alternatively, aggregation of the particulate solid phase in the reaction mixture can be measured by measuring the signal generated by the signal-forming system when the first and second components of the signal-forming system are in a spatially proximate state. In this regard, the first fraction of the particulate solid phase is associated with the first component of the signal-forming system, the second fraction of the particulate solid phase is associated with the second component of the signal-forming system, and the first and second components of the signal-forming system cooperate such that a detectable signal is generated when the first and second components of the signal-forming system are in a spatially proximate state, and the aggregation of the particulate solid phase in the reaction mixture is measured based on the generated signal.

[0055] In this embodiment of the method according to the invention, the signal-forming system comprises at least a first and a second component, which are in spatially close proximity and cooperate such that a detectable signal is generated when they are able to interact with each other. The interaction between the two components is understood to mean in particular energy transfer, i.e. the direct transfer of energy between the two components, for example through irradiation with light or electrons or via reactive chemical molecules such as singlet oxygen with a short lifetime. The energy transfer may be from one component to the other, but cascades of various substances through which the energy transfer takes place are also conceivable. For example, the two components can be a pair comprising an energy donor and an energy acceptor, for example a photosensitizer and a chemiluminescent agent (EP-A2-0515194, LOCI® Technologie) or a photosensitizer and a phosphor (WO95 / 06877) or radioactive iodine-125 and a phosphor (Udenfriend et al. (1985) Proc. Natl. Acad. Sci. 82: 8672-8676) or a phosphor and a fluorescence quencher (US 3,996,345). Particularly preferably, the first component of the signal-forming system is a chemiluminescent agent and the second component of the signal-forming system is a photosensitizer or vice versa, and the chemiluminescence in the reaction mixture is measured.

[0056] The following examples and figures are intended to illustrate the invention and should not be construed as limiting.

Examples

[0057] Example 1: Latex agglutination assay for the determination of VWF activity according to the prior art Reagent 1: HBR-1 reagent (heterophilic blocking reagent 1, Scantibodies Laboratory, Inc., Santee, USA) containing a mixture of mouse immunoglobulins for binding to heterophilic antibodies

[0058] Reagent 2: Recombinant expressed GPIb protein fragment of a gain-of-function variant of human GPIb protein in buffer.

[0059] Reagent 3: Suspension of polystyrene particles (latex particles) coated with a mouse monoclonal anti-GPIb antibody.

[0060] The von Willebrand factor (VWF) activity in the plasma sample was determined as follows: 1. Mix 40 μL of the sample with 2 μL of Reagent 1, and incubate the thus-treated sample at room temperature for 30 minutes. 2. Then, mix 15 μL of the pretreated sample with 30 μL of Owren’s Veronal buffer, with 70 μL of a buffer containing an additional surfactant, and with 15 μL of Reagent 2, and incubate the mixture at 37 °C for 2 minutes. 3. Then, add 40 μL of Reagent 3 to the mixture, and measure the change in absorbance of the reaction mixture with light at a wavelength of 570 nm. 4. Evaluate the measured raw values using a calibration curve.

[0061] Despite the use of the HBR-1 reagent, samples are sometimes present in which false high results are obtained due to the clearly insufficient blocking of HAMA intended by Reagent 1.

[0062] Example 2: Generation of a variant of an anti-GPIb antibody having reduced GPIb-binding ability The complete amino acid sequence of the mouse monoclonal anti-GPIb antibody used as the analytical antibody in the VWF assay according to Example 1 was determined.

[0063] Complementary determining region

Chemical formula

Chemical formula

[0064] The variant of the anti-GPIb antibody having reduced GPIb-binding ability was generated by substitution of the amino acid residues at positions 105 and 107 of SEQ ID NO: 3 in the CDR-H3 region. At position 105, the relatively large amino acid tyrosine (Y) was replaced by the very small and short amino acid serine (S) (bold font in the two sequences shown). At position 107, methionine (M) was replaced by phenylalanine (F), which corresponds to a reverse mutation to the mouse antibody germline (bold font in the two sequences shown). Appropriately encoded nucleic acid molecules were derived, and transgenic expression cell lines expressing the modified antibody were established using standard genetic engineering methods. However, the amino acid sequence of the modified antibody is identical to that of the anti-GPIb antibody except for the two modified amino acid residues described above.

[0065] Therefore, the complementarity-determining region

Chemical formula

Chemical formula

[0066] Example 3: Detection of reduced GPIb-binding ability of a novel antibody variant The assay system used was a latex agglutination assay for the determination of VWF activity according to Example 1.

[0067] Instead of reagent 1 (HBR-1 reagent), the assay was modified such that in each case, 2 μL of reagent containing various amounts of the novel antibody variant generated according to Example 2 was mixed and incubated with 40 μL of a normal plasma sample or a sample known to have reduced VWF activity.

[0068] The results are shown in Figure 1A. At final concentrations up to 0.02 mg / mL of the novel antibody variant in the final reaction mixture, the measured VWF activity was reduced by up to 2.3% compared to the reaction mixture without the addition of the novel antibody variant (0 mg / mL). This indicates that the novel antibody variant does not compete with the functional (''analytical'') mouse monoclonal anti-GPIb antibody for the binding site on the GPIb protein.

[0069] The novel antibody variant generated according to Example 2 has an amino acid sequence that is identical to that of the functional anti-GPIb antibody except for the aforementioned two modified amino acid residues, but as a result, has a significantly reduced GPIb binding ability compared to the functional anti-GPIb antibody.

[0070] For comparison, the assay was modified in terms of additional variants such that 2 μL of various dilutions of reagent 1 (HBR-1 reagent) containing various total protein concentrations were mixed and incubated with 40 μL of normal plasma sample or 40 μL of a sample known to have reduced VWF activity in each case.

[0071] The results are shown in Figure 1B. At final concentrations up to 0.02 mg / mL of the HBR total protein in the final reaction mixture, the measured VWF activity was reduced by up to 2.6% compared to the reaction mixture without the addition of the HBR-1 reagent (0 mg / mL). This observation supports the conclusion that the reduction in VWF activity caused by the novel antibody variant is not a specific effect of the antibody variant.

[0072] Example 4: Detection of the HAMA antibody blocking effect of the novel antibody variant Instead of reagent 1 (HBR-1 reagent), 2 μL of a reagent containing various amounts of the novel antibody variant generated according to Example 2 alone, or 2 μL of a reagent containing various amounts of the novel antibody variant generated according to Example 2 in combination with reagent 1 (HBR-1 reagent), in each case, was mixed with 40 μL of a HAMA antibody-containing plasma sample having known VWF activity and incubated, modifying the latex agglutination assay for the determination of VWF activity according to Example 1. The samples used were identified by the fact that they could not be sufficiently blocked by the sole use of the HBR-1 reagent, resulting in an erroneously high VWF activity being determined.

[0073] The results are shown in Figure 2. At final concentrations from 0.005 mg / mL for the novel antibody variant in the final reaction mixture, which can already be observed, is an almost complete blocking of the interfering effect of the HAMA antibody. In contrast, the sole use of the HBR-1 reagent only results in an insufficient blocking of the interfering effect of the HAMA antibody. The combination of the novel antibody variant with the HBR-1 reagent did not show a blocking effect that exceeded the blocking effect of the novel antibody variant, or a blocking effect indicating that the blocking effect was impaired.

Claims

**Claim 1** A kit for use in a method of detecting an analyte in a body fluid sample, comprising: a) a first antigen-specific antibody having a first amino acid sequence, specifically binding to an antigen, wherein its use results in an analyte-specific detection reaction in a defined assay system for detecting the analyte; b) an antibody variant having a second amino acid sequence, wherein the antigen-binding ability of the antibody variant is significantly reduced compared to the first antibody, such that the additional use of the antibody variant in a defined assay system for detecting the analyte results in a maximum 15% reduction in the analyte-specific detection reaction; and the amino acid sequence of the antibody variant is identical to the amino acid sequence of the first antigen-specific antibody, except for 1 to 3 modified amino acid residues. A kit as claimed in claim 1. **Claim 2** The kit according to claim 1, wherein the 1 to 3 modified amino acid residues are located in one or more of the complementarity-determining regions (CDRs) of the heavy or light chain of the antibody variant. **Claim 3** The kit according to claim 2, wherein at least one modified amino acid residue is located in one complementarity-determining region of the heavy chain of the antibody variant. **Claim 4** The kit according to claim 3, wherein at least one modified amino acid residue is located in the complementarity-determining region CDR-H3 of the heavy chain of the antibody variant. **Claim 5** The kit according to any one of claims 1 to 4, wherein at least the antibody variant is recombinantly produced. **Claim 6** The kit according to any one of claims 1 to 5, wherein the first antigen-specific antibody and the antibody variant are present in different reagents. **Claim 7** The kit according to any one of claims 1 to 6, wherein the first antigen-specific antibody is associated with a solid phase and / or a component of a signal-forming system. **Claim 8** The kit according to any one of claims 1 to 7, wherein the first antigen-specific antibody is an analyte-specific antibody. **Claim 9** c) at least one further antigen-specific antibody having a third amino acid sequence, specifically binding to an antigen, wherein its use results in an analyte-specific detection reaction in a defined assay system for detecting the analyte; at least one further antigen-specific antibody; and d) a further antibody variant having a fourth amino acid sequence, The antigen-binding ability of the further antibody variant is significantly reduced compared to the further antigen-specific antibody, such that the additional use of the further antibody variant in a defined assay system for the detection of an analyte results in a maximum 15% reduction of the analyte-specific detection reaction, and the further antibody variant further comprises The amino acid sequence of the further antibody variant is identical to the amino acid sequence of the further antigen-specific antibody, except for 1 to 3 modified amino acid residues The kit according to any one of claims 1 to 8

10. The kit according to claim 1, wherein the first antigen-specific antibody specifically binds to the GPIb protein

11. The kit according to claim 10, wherein the first GPIb protein-specific antibody has an amino acid sequence according to SEQ ID NO: 1 in the complementarity-determining region CDR-H3 of the heavy chain

12. The kit according to claim 11, wherein the amino acid sequence of the antibody variant is identical to the amino acid sequence of the first GPIb protein-specific antibody, except for 2 modified amino acid residues, and has an amino acid sequence according to SEQ ID NO: 2 in the complementarity-determining region CDR-H3 of the heavy chain

13. Use of the kit according to any one of claims 1 to 12 in a method for detecting an analyte in a body fluid sample

14. Use of the kit according to any one of claims 1 to 15 for the interference-free detection of an analyte in a body fluid sample containing interfering antibodies, for example from the group of heterophilic and autoantibodies

15. Use of the kit according to any one of claims 10 to 12 in a method for detecting VWF activity in a body fluid sample

16. A method for detecting an analyte in a body fluid sample, comprising a) preparing a reaction mixture by mixing the sample with i. a first antigen-specific antibody, which has a first amino acid sequence, specifically binds to an antigen, and the use of which results in an analyte-specific detection reaction in a defined assay system for the detection of the analyte, the first antigen-specific antibody, and ii. an antibody variant, which has a second amino acid sequence, and the antigen-binding ability of which is significantly reduced compared to the first antibody, such that the additional use of the antibody variant in a defined assay system for the detection of the analyte results in a maximum 15% reduction of the analyte-specific detection reaction the antibody variant ​ b) measuring a measurement variable in the reaction mixture that is affected by the formation of a complex of the antigen and the first antigen-specific antibody and correlates with the amount of the analyte; comprising; The amino acid sequence of the antibody variant is identical to the amino acid sequence of the first antigen-specific antibody, except for 1 to 3 modified amino acid residues, method.

17. The method according to claim 16, wherein the sample is first mixed with the antibody variant, the resulting mixture is incubated, and then the first antigen-specific antibody is added to the mixture.

18. The method according to claim 16 or 17, wherein the first antigen-specific antibody is a specific antibody for the analyte, and the measurement variable to be measured is affected by the formation of a complex of the analyte and the first analyte-specific antibody.

19. The method according to claim 16 or 17, wherein the first antigen-specific antibody is an antibody having specificity for a binding partner of the analyte, and the measurement variable to be measured is affected by the formation of a complex of the analyte, the binding partner of the analyte, and the first antigen-specific antibody having specificity for the binding partner of the analyte.

20. The first antigen-specific antibody is an antibody having specificity for the GPIb protein, The measurement variable to be measured is affected by the formation of a complex of von Willebrand factor, the GPIb protein, and the first antigen-specific antibody having specificity for the GPIb protein, The method according to claim 19 for detecting the activity of von Willebrand factor in a body fluid sample.

21. The first antigen-specific antibody is associated with a particulate solid phase, and the aggregation of the particulate solid phase in the reaction mixture, which is affected by the formation of a complex of the antigen and the first antigen-specific antibody and correlates with the amount of the analyte, is measured, The method according to any one of claims 16 to 20.

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