Methods and kits for reducing interference in immunoassays

Abstract

The present invention relates to methods and kits for reducing interference in immunoassays. In particular, the present invention belongs to the field of immunoassays for in vitro diagnostic applications and relates to the use of testing specific blocking antibodies for reducing interference caused by heterophilic antibodies or rheumatoid factors.

Description

Technical Field

[0001] The present invention belongs to the field of immunoassays for in vitro diagnostic applications and relates to the use of test specific blocking antibodies to reduce interference caused by heterophilic antibodies or rheumatoid factors. Background Art

[0002] For decades, immunoassays have been used in clinical diagnostic tests to quantitatively or qualitatively detect various analytes in body fluid samples. Antibodies for direct antigen detection or for indirect detection of different analytes are usually monoclonal or polyclonal animal antibodies obtained from immunized animals (such as rabbits, mice, sheep) or by biotechnological means. False test results caused by interfering substances in patient samples can lead to far-reaching misdiagnosis. A known and relatively frequent problem is the possible presence of interfering antibodies in individual samples (Bolstad, N. et al., Heterophilic antibody interference in immunometric assays. Best Practice & Research Clinical Endocrinology & Metabolism (2013), 647 - 661). An example of an interfering antibody is a heterophilic antibody, i.e., an antibody in a patient's blood against antigens of other species, especially immunoglobulins, such as human anti - mouse antibody (HAMA). Another example of an interfering antibody is the so - called rheumatoid factor, i.e., a human auto - antibody against the Fc portion of human immunoglobulin G. All these interfering antibodies usually have an affinity for animal antibodies and often bind to the Fc portion. Then, if the test system uses an animal antibody as part of the detection reaction for detecting an analyte ("analytical antibody"), in the presence of interfering antibodies from a patient sample, it is possible that a binding reaction occurs between the analytical antibody and the interfering antibody, which either blocks the detection reaction and results in false negative / false low results, or enhances the detection reaction and results in false positive / false high results.

[0003] Thus, to reduce such interference, the standard procedure is to add blocking antibodies to modern immunoassays. The blocking antibodies are typically a mixture of randomly selected non-specific antibodies that belong to the same immunoglobulin class and species as the analyte-specific antibodies used in the test system. For example, in a test system using a monoclonal mouse antibody (e.g., mouse IgG1) as the analyte-specific antibody, an additional additive can be a mixture of excess irrelevant mouse IgG1, i.e., mouse IgG1 that does not function in the test system. In most cases, this causes any interfering antibodies from the patient sample to bind to the irrelevant antibodies, thus preventing or at least minimizing the disruptive binding to the analyte-specific antibody. Such blocking antibodies are commercially available, e.g., the heterophilic blocking reagent (HBR) from Scantibodies Laboratory, Inc. or the TRU Blocking reagent from Meridian Bioscience, Inc. The blocking effect of such irrelevant antibodies can be optimized by pre-aggregation of the irrelevant antibodies (US2004 / 0018556 A1).

[0004] Despite these measures, in some cases, due to the demonstrably insufficiently effective blocking by conventional blocking reagents, certain samples cannot be properly analyzed using a specific immunoassay test system. Bowyer A.E. et al. (VonWillebrand factor activity assay errors. Haemophilia (2016), 22, e74-e76) described patient samples assayed for von Willebrand factor (VWF) activity using two different test systems, involving the use of in particular monoclonal mouse antibodies in both cases, which repeatedly gave false high results despite the addition of a HAMA blocker (for blocking human anti-mouse antibodies).

[0005] An "immunoassay" in the context of the present invention is a method for detecting an analyte in a sample that comprises using at least one antigen-specific antibody. The antigen-specific antibody can be (but is not necessarily) an analyte-specific antibody.

[0006] Depending on the test setup, the antibodies used can perform multiple functions. For example, it can be used as a capture antibody or a labeled secondary antibody for direct binding and detection of the analyte; for this purpose, the antibody must be an analyte-specific antibody. In another case, an antibody can be used, for example, to immobilize a binding partner of the analyte to be detected on a solid phase; for this purpose, the antibody must be an antibody specific for the said binding partner. There are various immunoassay principles known (direct, indirect, competitive, non-competitive). All have in common that they involve the use of at least one antigen-specific antibody which directly or indirectly participates in the analyte-specific detection reaction in a selected test system for detecting the analyte.

[0007] Accordingly, a conventional immunoassay optimized to minimize the occurrence of antibody-induced interference is a method for detecting an analyte in a body fluid sample, which essentially comprises the steps of:

[0008] i) providing a reaction mixture by contacting the sample with a) and b):

[0009] a) an antigen-specific antibody that binds specifically to the antigen, and

[0010] 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, and

[0011] ii) measuring a measurement variable in the reaction mixture that is affected by the formation of a complex of the antigen with the first antigen-specific antibody and is related to the amount of the analyte.

[0012] Since there are always some samples that cannot be correctly analyzed in certain immunoassays due to insufficiently effective blocking despite the addition of blocking antibodies to the reaction mixture, 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 interfering antibodies (such as heterophilic antibodies or rheumatoid factors) present in patient samples. Summary of the Invention

[0013] The object is achieved according to the invention by using a blocking antibody derived from an analytical antibody and having a structure almost identical to that of the analytical antibody. Thus, any interfering antibody from a patient sample that binds highly specifically to the epitope of the analytical antibody and is thus not blocked or is insufficiently blocked by a classical randomly selected mixture of blocking antibodies is now specifically captured and thus blocked by binding to the derived blocking antibody. It has been found that an antibody having an amino acid sequence identical to that of the analytical antibody except for one to three modified amino acid residues and thus having a greatly reduced antigen-binding capacity compared to the analytical antibody results in efficient blocking of interfering antibodies and thus significantly improves the reliability of immunoassays.

[0014] Accordingly, the invention provides a kit for use in a method for detecting an analyte in a body fluid sample. The kit contains:

[0015] i) a first antigen-specific antibody having a first amino acid sequence, which binds specifically to an antigen and whose use in a defined test system for analyte detection gives rise to an analyte-specific detection reaction ("analytical antibody"), and

[0016] ii) an antibody variant having a second amino acid sequence, wherein the antigen-binding capacity of the antibody variant is greatly reduced compared to the first antibody, or its competition with the first antigen-specific antibody for binding to the antigen is so low that its additional use in a defined test system for analyte detection reduces the analyte-specific detection reaction by at most 15% (also referred to as "non-analytical antibody" or "non-analytical antibody variant"),

[0017] and wherein the amino acid sequence of the antibody variant is identical to that of the first antigen-specific antibody except for one to three modified amino acid residues.

[0018] A kit for use in a method for detecting an analyte in a body fluid sample generally contains one or more reagents in liquid or lyophilized form or in coated solid phase form, which are brought into contact with the body fluid sample to be analyzed (e.g., whole blood, plasma, serum, urine) to produce a detection reaction, thereby allowing a quantitative, semi-quantitative or qualitative determination of the amount or activity of the analyte.

[0019] The first antigen-specific antibody binds specifically to the antigen and is essential for generating the expected analyte-specific detection reaction of the test system ("analytical antibody"). The antigen-specific antibody can be an analyte-specific antibody that binds specifically to the analyte from a body fluid sample. Alternatively, the antibody can be an antibody that binds specifically to a binding partner of the analyte. In this case, the binding partner of the analyte can be a binding partner that inherently exists in the body fluid sample or can be a binding partner added to the reaction mixture. In another embodiment of the detection method, the antibody can be an antibody that binds specifically to a cleavage product of the analyte.

[0020] Depending on the test setup, the antibody used may perform multiple functions. For example, it can be used as a capture antibody or a labeled secondary antibody for directly binding to and detecting the analyte; for this purpose, the antibody must be an analyte-specific antibody. In another case, an antibody can be used, for example, to immobilize a binding partner of the analyte to be detected on a solid phase; for this purpose, the antibody must be specific for the said binding partner. There are various immunoassay principles (direct, indirect, competitive, non-competitive). All have in common that they involve the use of at least one antigen-specific antibody that directly or indirectly participates in the analyte-specific detection reaction in a selected test system for detecting the analyte.

[0021] The first antigen-specific antibody can belong to any immunoglobulin class (IgA, IgD, IgE, IgG or IgM); its original source can be human, mouse, rabbit, sheep, camel or other animals. Preferably, the antibody is a monoclonal antibody or a recombinantly produced antibody. The first antigen-specific antibody can also be a chimeric antibody or a humanized antibody. The term "first antigen-specific antibody" clearly includes not only whole antibodies but also various antigen-binding antibody fragments, such as Fab or F(ab)2 fragments.

[0022] In various embodiments, the first antigen-specific antibody can be associated with a solid phase and / or components of a signal-forming system.

[0023] In the context of the present invention, the term "solid phase" includes objects composed of porous and / or non-porous, water-insoluble materials, and it can take various forms, such as containers, tubes, microtiter plates (ELISA plates), beads, microparticles, rods, strips, filter paper or chromatography paper, etc. 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, naturally occurring materials and / or modified naturally occurring 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 precious metals such as gold and silver; magnetite; their mixtures or combinations. Particles (including magnetic particles and latex particles) can be labeled with dyes, sensitizers, fluorescent substances, chemiluminescent substances, isotopes or other detectable markers.

[0024] "Components of the signal-forming system" refer to molecules that can generate signals by themselves or can induce the generation of signals, such as fluorescent substances, chemiluminescent substances, radioactive substances or enzymes. For example, signals can be detected or measured based on enzyme activity, luminescence, light absorption, light scattering, emitted electromagnetic or radioactive radiation or chemical reactions.

[0025] Suitable components of the signal-forming system are, for example, enzymes, including horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, glucose oxidase, β-galactosidase, luciferase, urease and acetylcholinesterase; enzyme substrates; dyes; fluorescent substances, including fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, ethidium bromide, 5-dimethylaminonaphthalene-1-sulfonyl chloride and fluorescent chelates of rare earths; chemiluminescent substances, including luminol, isoluminol, acridine compounds, olefins, enol ethers, enamines, aryl vinyl ethers, dioxenes, aryl imidazoles, lucigenin, fluorescein and luminescent proteins (aequorin); sensitizers, including eosin, 9,10-dibromoanthracene, methylene blue, porphyrins, phthalocyanines, chlorophyll, rose bengal; coenzymes; radioactive isotopes, including 125 I, 131 I, 14 C, 3 H, 32 P, 33 P, 35 S, 51 Cr, 59 Fe, 57 Co and 75 Se.

[0026] The term "associated" should be understood in a broad sense and includes, for example, covalent and non-covalent bonds, direct and indirect binding, adsorption to a surface, and inclusion in a recess. In the case of a covalent bond, the first antigen-specific antibody or antigen-specific antibody fragment is bound to a component of the solid phase or the signal-forming system by 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 by specific interaction with other binding partners. For example, if the first antigen-specific antibody or antigen-specific antibody fragment is biotinylated, it binds by specific interaction with avidin.

[0027] An antibody variant of the kit according to the invention is an antibody (also referred to as "non-analytical antibody" or "non-analytical antibody variant") derived from the first antigen-specific antibody or the corresponding antibody fragment, and has 1.) an amino acid sequence which is identical to the amino acid sequence of the first antigen-specific antibody except for one to three modified amino acid residues, and 2.) an antigen-binding ability which is significantly reduced compared to the first antigen-specific antibody, such that its additional use in a defined test system for detecting an analyte reduces the analyte-specific detection reaction by at most 15%.

[0028] Thus, such a "non-analytical" antibody variant is tailored to the first antigen-specific antibody ("analytical antibody") alone and can generally be obtained by defining a modified amino acid sequence by substitution, deletion, insertion or chemical derivatization of one to three amino acid residues from the known amino acid sequence of the analytical antibody (or after the amino acid sequence of the analytical antibody has been determined), and recombinantly producing the corresponding modified antibody variant. The position of one modified amino acid residue or the positions of the two or three modified amino acid residues are selected such that they are located in a region of the analytical antibody which is related to antigen binding and will be functionally restricted or inactivated by this modification of the amino acid sequence, such that the antigen-binding ability of the resulting modified non-analytical antibody variant is absent or at least only significantly reduced. For this purpose, it is preferred to modify (i.e., substitute, delete, insert or chemically derivatize) the one to three amino acid residues in one or more of the complementarity-determining regions (CDRs) of the heavy or light chain of the analytical antibody. The complementarity-determining regions (CDRs) of the antibody heavy chain (CDR-H1, CDR-H2 and CDR-H3) and the complementarity-determining regions (CDRs) of the antibody light chain (CDR-L1, CDR-L2 and CDR-L3) (according to the Kabat numbering scheme), which are separated from each other by so-called framework regions, are well known to the person skilled in the art. Particularly preferably, at least one amino acid residue in the complementarity-determining region of the heavy chain of the analytical antibody is modified. More preferably, at least one amino acid residue in the complementarity-determining region CDR-H3 of the heavy chain of the analytical antibody is modified.

[0029] 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.

[0030] 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 a complementarity determining region of the heavy chain of the antibody variant.

[0031] In yet a further embodiment of the test kit, there is an antibody variant in which at least one modified amino acid residue is located in complementarity determining region CDR-H3 of the heavy chain of the antibody variant.

[0032] A "modified amino acid residue" is to be understood as an amino acid residue that is related to a position in the amino acid sequence of the analytical antibody primary sequence and is substituted, deleted, inserted or chemically derivatized. In the case of a 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 amino acid families that differ in side chain 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 a charged amino acid residue.

[0033] A "non-analytical" antibody variant can also be a natural variant of the first antigen-specific antibody, in which the amino acid sequence of the variant has one to three substituted, deleted or inserted amino acid residues compared to the original analytical antibody.

[0034] Thus, compared to the first antigen-specific antibody, the antibody variant is a variant that is non-functional or at least less functional in terms of antigen-binding ability.

[0035] Compared to the first antigen-specific antibody, the antigen-binding ability of the antibody variant must be significantly reduced such that its additional use in a defined test system for analyte detection reduces the analyte-specific detection reaction by at most 15%.

[0036] The reduced antigen-binding ability of the antibody variant can be measured in a standard assay for determining the binding specificity of the target antigen, such as in an ELISA assay, a BIAcore assay, an Octet BLI assay, or a FACS-based assay (if the antigen is expressed on the cell surface), in a comparative experiment using the first antigen-specific antibody.

[0037] However, it is crucial to test the antigen-binding ability of antibody variants in the same defined test system for analyte detection, where the first antigen-specific antibody is used as the "analytical" antibody. The term "defined test system" refers to a test setup that is defined with respect to the components and method steps used. Changes in individual components or individual method steps in a test setup that is otherwise kept constant allow the determination of the impact of such changes in the test system defined in that other respect. Ideally, the method for detecting the analyte is used as the defined test system, where the first antigen-specific antibody and the antibody variant are intended to be used to achieve it. For the purposes of the present invention, the first antigen-specific antibody for detecting the analyte is used, and additionally (i.e., in combination with the first antigen-specific antibody) the antibody variant to be tested.

[0038] In a test system where the first antigen-specific antibody is used as the "analytical" antibody, a suitable antibody variant has no significant competitive effect on the specific detection reaction of the analyte. First, this is ensured by measuring the reaction intensity of the analyte-specific detection reaction over the entire measurement range in the test system, with and without the addition of the antibody variant, in a sample that does not have interfering antibodies. A suitable antibody variant is an antibody variant with antigen-binding ability, the antigen-binding ability of which is significantly reduced compared to the first antigen-specific antibody such that its presence reduces the reaction intensity of the analyte-specific detection reaction by no more than 15%, preferably no more than 10% and particularly preferably no more than 5%; by demonstrating that the combined use of the antibody variant with the first antigen-specific antibody does not have an excessive disruptive effect on the analyte-specific detection reaction, but rather reduces the reaction intensity of the analyte-specific detection reaction by a maximum of 15%, it is functionally demonstrated that there is little or no competition between the antibody variant and the first antigen-specific antibody for antigen binding, i.e., little or no competition for the binding site of the antigen.

[0039] In a kit according to the present invention, the first antigen-specific antibody and the antibody variant can be present in different reagents or in a single reagent.

[0040] 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), such as at the bottom of a well of a microtiter plate or inside a reaction tube. Such kits are particularly suitable for performing heterogeneous test methods, such as ELISA tests. Such kits preferably also contain a further container that contains the antibody variant, preferably as a component of a liquid reagent (or a component of the lyophilized product of a liquid reagent).

[0041] In another embodiment of the kit, the first antigen-specific antibody is associated with the surface of the particulate solid phase (as described above). To this end, the kit contains a container that contains the corresponding reagent in the form of a liquid suspension or its resuspendable lyophilizate. Such test kits are suitable for measuring agglutination by photometry.

[0042] 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 can be directly associated with a component of the signal-forming system or indirectly associated, for example when both the antibody and the component of the signal-forming system are associated with a single solid phase (such as latex particles). To this end, the kit contains a container that contains the corresponding reagent in liquid form or as its resuspendable lyophilizate. Depending on the nature of the signal-forming system, such test kits are suitable for measuring, for example, chemiluminescence, fluorescence or absorption changes.

[0043] In a particularly preferred kit, the first antigen-specific antibody is an analyte-specific antibody. In this case, the antibody is directly used as a capture antibody or a labeled secondary antibody for binding and detecting the analyte, for example in a sandwich immunoassay.

[0044] In another embodiment of the kit, in addition to the first antigen-specific antibody, a different second antigen-specific antibody is additionally present, for example in a kit for a sandwich immunoassay, where the second antigen-specific antibody can be specific for the same antigen as the first antigen-specific antibody or for a different antigen. In such kits, a further "non-analytical" antibody variant (as described above) is preferably present, which is a non-functional or at least less functional variant with respect to antigen-binding ability compared to the second antigen-specific antibody. Thus, such kits additionally contain:

[0045] c) a second antigen-specific antibody having a third amino acid sequence that specifically binds the antigen and whose use in a defined test system for analyte detection gives rise to an analyte-specific detection reaction, and

[0046] d) an additional antibody variant having a fourth amino acid sequence and whose antigen-binding ability is substantially reduced compared to the second antigen-specific antibody such that its additional use in a defined test system for analyte detection reduces the analyte-specific detection reaction by at most 15%,

[0047] and wherein the amino acid sequence of the additional antibody variant is identical to the amino acid sequence of the second antigen-specific antibody except for one to three modified amino acid residues.

[0048] Preferably, in the kit according to the present invention, each antigen-specific antibody present is provided with an antibody variant which, compared to the corresponding antigen-specific antibody, is a non-functional or at least less functional variant with respect to antigen-binding ability (as described above).

[0049] The present invention further provides the use of the kit according to the present invention in a method for detecting an analyte in a body fluid sample.

[0050] Particularly preferably, the kit according to the present invention is used for interference-free detection of an analyte in a body fluid sample containing interfering antibodies, such as heterophilic antibodies and autoantibodies.

[0051] In a specific embodiment of the kit, the first antigen-specific antibody is an antibody that specifically binds glycoprotein Ib (GPIb). The GPIb protein is a binding partner of von Willebrand factor (VWF) and is used in various assays for determining VWF activity (see, for example, WO 2009 / 007051A2). A qualitative or functional defect of VWF is detected by a decrease in the binding of VWF present in the sample to the added GPIb protein. By configuring the test method such that the complex formed between VWF and GPIb in the test reaction can be measured, for example, by measuring the agglutination of latex particles, the ability of VWF to bind to the added GPIb protein can be quantitatively determined. The latex particles are coated with an anti-GPIb antibody and agglutinate only when the VWF-GPIb complex is formed in the test reaction and then becomes associated with the anti-GPIb antibody associated with the latex particles. As described above, it has been observed that such assays using a mouse monoclonal anti-GPIb antibody repeatedly produce false high results despite the addition of an HAMA blocker (for blocking human anti-mouse antibodies).

[0052] In a preferred form of the specific embodiment of the kit,

[0053] · the first antigen-specific antibody is an antibody that specifically binds glycoprotein Ib (GPIb) protein and has an amino acid sequence according to SEQ ID NO.1 (DTMIKGHYVMDY) in the complementarity determining region CDR-H3 of the heavy chain (according to the Kabat numbering scheme), and

[0054] · the antibody variant is an antibody that, except for two modified amino acid residues, has the same amino acid sequence as the first GPIb protein-specific antibody and has an amino acid sequence according to SEQ ID NO.2 (DTMIKGHSVFDY) in the complementarity determining region CDR-H3 of the heavy chain (according to the Kabat numbering scheme).

[0055] This test kit is suitable for use in a method for detecting the activity of VWF in a body fluid sample and has the particular advantage of allowing interference-free detection of the VWF activity in a body fluid sample containing interfering antibodies, such as heterophilic antibodies and autoantibodies.

[0056] The present invention further provides a method for detecting an analyte in a body fluid sample, the method comprising the steps of:

[0057] a) providing a reaction mixture by mixing the sample with i and ii:

[0058] i. a first antigen-specific antibody, which has a first amino acid sequence, specifically binds to an antigen, and whose use in a defined test system for analyte detection causes an analyte-specific detection reaction, and

[0059] ii. an antibody variant, which has a second amino acid sequence and whose antigen-binding ability is significantly reduced compared to the first antibody such that its additional use in a defined test system for analyte detection reduces the analyte-specific detection reaction by at most 15%; and

[0060] b) measuring a measurement variable in the reaction mixture, which is affected by the formation of a complex between the antigen and the first antigen-specific antibody and is related to the amount of analyte,

[0061] wherein, here too (as described above), the amino acid sequence of the antibody variant is identical to the amino acid sequence of the first antigen-specific antibody except for one to three modified amino acid residues.

[0062] Preferably, 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. This pre-incubation of the sample with the "non-analyte" antibody variant particularly effectively blocks interfering antibodies because they are bound before coming into contact with the "analyte" antibody.

[0063] In one embodiment of the method according to the invention, the first antigen-specific antibody is an analyte-specific antibody and the measured measurement variable 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 a complex of the analyte and the analyte-specific antibody associated with latex particles is formed and the formation of the complex is determined photometrically based on the agglutination reaction of the latex particles in the reaction mixture.

[0064] In another embodiment of the method according to the invention, the first antigen-specific antibody is an antibody specific for the binding partner of the analyte, and the measured measurement variable is affected by the formation of a complex of the analyte, the binding partner of the analyte and the first antigen-specific antibody specific for the binding partner of the analyte. An example thereof is a functional binding test, by which the amount of the analyte is not measured, but the ability of the analyte to bind specifically to the binding partner is measured, and in which a complex of the analyte, the binding partner of the analyte and an antibody specific for the binding partner, for example associated with latex particles, is formed, and the formation of the complex is determined photometrically based on the agglutination reaction of the latex particles in the reaction mixture.

[0065] 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, wherein the first antigen-specific antibody is an antibody specific for the GPIb protein, and wherein the measured measurement variable is affected by the formation of a complex of von Willebrand factor, the GPIb protein and the first antigen-specific antibody specific for the GPIb protein.

[0066] In the method according to the invention, the first antigen-specific antibody can be associated with a particulate solid phase, and the agglutination of the particulate solid phase in the reaction mixture can be measured, which agglutination is affected by the formation of a complex of the antigen and the first antigen-specific antibody and is related to the amount of the analyte.

[0067] The agglutination of the particulate solid phase in the reaction mixture can be measured photometrically (e.g., nephelometry or turbidimetry). Since about 1920, binding tests based on the principle of particle-enhanced light scattering have been known (for a review, see Newman, D.J. et al., Particle enhanced light scattering immunoassay. Ann Clin Biochem 1992; 29: 22-42). In this case, polystyrene particles with a diameter of 0.1 to 0.5 μm, more preferably with a diameter of 0.15 to 0.35 μm, are preferably considered. Polystyrene particles with amine, carboxyl or aldehyde functional groups are preferably considered. The use of core and shell particles is also preferably considered. For example, the synthesis of the particles and the covalent coupling of the ligands are described in 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.

[0068] Alternatively, when the first and second components of the signal-forming system come into spatial proximity, the agglutination of the particulate solid phase in the reaction mixture can be measured by measuring the signal generated by the signal-forming system. In this context, the first fraction of the particulate solid phase is associated with the first component of the signal-forming system, and 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 come into spatial proximity, and the agglutination of the particulate solid phase in the reaction mixture is measured based on the generated signal.

[0069] In this embodiment of the method according to the invention, the signal-forming system comprises at least a first component and a second component, which components assist in generating a detectable signal when they come into spatial proximity and can thus interact with each other. The interaction between the components should be understood to refer in particular to energy transfer, i.e. the direct transfer of energy between the components, for example by light or electron irradiation or by reactive chemical molecules such as short-lived singlet oxygen. Energy transfer can occur from one component to another, but cascades of energy transfer via different substances are also possible. For example, the components can be a pair comprising an energy donor and an energy acceptor, such as a photosensitizer and a chemiluminescent agent (EP-A2-0515194, Technologie) or a photosensitizer and a fluorophore (WO 95 / 06877) or radioactive iodine-125 and a fluorophore (Udenfriend et al. (1985) Proc. Natl. Acad. Sci. 82:8672-8676) or a fluorophore 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.

[0070] The following examples and figures are used to illustrate the invention and should not be construed as limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1a Charts are shown which show the VWF activity [% normal] measured in normal samples (1) and samples with reduced VWF activity (2) in the absence or presence of different concentrations (mg / mL) of the new antibody variant in the reaction mixture.

[0072] Figure 1b Charts are shown which show the VWF activity [% normal] measured in normal samples (3) and samples with reduced VWF activity (4) using different dilutions of the commercial HBR-1 reagent (total protein content in mg / mL in the reaction mixture).

[0073] Figure 2 Disclosed is a graph showing the VWF activity [normal %] measured in a sample containing HAMA antibodies, additionally using different dilutions of a commercial HBR-1 reagent (total protein content in mg / mL in the reaction mixture) (1), additionally using different concentrations of a new antibody variant (mg / mL) in the reaction mixture (2), or additionally using different concentrations of a new antibody variant (mg / mL) and the HBR-1 reagent in the reaction mixture (3). Detailed Description

[0074] Examples

[0075] Example 1: Latex agglutination assay for determining VWF activity according to the prior art

[0076] Reagent 1:

[0077] The HBR-1 reagent (heterophilic blocking reagent 1, Scantibodies Laboratory, Inc., Santee, USA), contains a mixture of murine immunoglobulins for binding to heterophilic antibodies.

[0078] Reagent 2:

[0079] In a buffer solution, a recombinant expressed GPIb protein fragment of a gain-of-function mutant of human GPIb protein.

[0080] Reagent 3:

[0081] A suspension of polystyrene particles (latex particles) coated with a murine monoclonal anti-GPIb antibody.

[0082] The von Willebrand factor (VWF) activity in a plasma sample was determined as follows:

[0083] 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.

[0084] 2. Then, mix 15 μL of the pre-treated sample with 30 μL of Owren's Veronal Buffer, 70 μL of a buffer containing additional detergent, and 15 μL of reagent 2 and incubate the mixture at 37 °C for 2 minutes.

[0085] 3. Then, add 40 μL of reagent 3 to the mixture and measure the change in absorbance of the reaction mixture with light of a wavelength of 570 nm.

[0086] 4. Evaluate the measured raw values with the aid of a calibration curve.

[0087] Although the HBR-1 reagent was used, since Reagent 1 was not significantly effective enough in blocking HAMA, false high results were occasionally obtained for some samples.

[0088] Example 2: Production of variants of anti-GPIB antibodies with reduced GPIb-binding ability

[0089] The complete amino acid sequence of the murine monoclonal anti-GPIb antibody used as the analytical antibody in the VWF assay according to Example 1 was determined.

[0090] Comprising complementarity determining regions and CDR-H3 The amino acid sequence in the heavy variable chain region of said anti-GPIb antibody (according to the Kabat numbering scheme) is as follows:

[0091]

[0092]

[0093] Variants of said anti-GPIb antibody with reduced GPIb-binding ability were generated by substituting 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) (shown in bold in the two sequences). At position 107, methionine (M) was replaced by phenylalanine (F), which corresponds to a reversal to the murine antibody germline (shown in bold in the two sequences). Appropriately encoded nucleic acid molecules were derived and transgenic expression cell lines expressing the modified antibody were established using standard genetic engineering methods. 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.

[0094] Thus, the amino acid sequence in the heavy variable chain region of said modified antibody comprising complementarity determining regions and CDR-H3 is as follows:

[0095]

[0096] Example 3: Detection of reduced GPIb-binding ability of the new antibody variant

[0097] The test system used was a latex agglutination assay for determining VWF activity according to Example 1.

[0098] The assay was modified such that instead of reagent 1 (HBR-1 reagent), in each case, 2 μL of a reagent containing different amounts of the new antibody variants produced according to the examples was mixed and incubated with 40 μL of a normal plasma sample or a sample with a known reduced VWF activity.

[0099] Figure 1a The results are shown. When the final concentration of the new antibody variant in the final reaction mixture was up to 0.02 mg / mL, the measured VWF activity decreased by a maximum of 2.3% compared to the reaction mixture without the addition of the new antibody variant (0 mg / mL). This indicates that the new antibody variant does not compete with the functional (“analytical”) mouse monoclonal anti-GPIb antibody for the binding site on the GPIb protein.

[0100] The new antibody variant produced according to Example 2 has an amino acid sequence identical to that of the functional anti-GPIb antibody except for the two modified amino acid residues mentioned above, and thus has a significantly reduced GPIb-binding ability compared to the functional anti-GPIb antibody.

[0101] For comparison purposes, in a further variant, the assay was modified such that in each case, 2 μL of different dilutions of reagent 1 (HBR-1 reagent) containing different total protein concentrations was mixed and incubated with 40 μL of a normal plasma sample or a sample with a known reduced VWF activity.

[0102] Figure 1b The results are shown. When the final concentration of the total HBR protein in the final reaction mixture was up to 0.02 mg / mL, the measured VWF activity decreased by a maximum of 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 decrease in VWF activity caused by the new antibody variant is not a specific effect of the antibody variant.

[0103] Example 4: Detection of the HAMA antibody blocking effect of the new antibody variant

[0104] The latex agglutination assay for determining VWF activity according to Example 1 was modified such that instead of reagent 1 (HBR-1 reagent), in each case, 2 μL of a reagent containing different amounts of the new antibody variant produced according to Example 2, or containing different amounts of the new antibody variant produced according to Example 2 and reagent 1 (HBR-1 reagent), was mixed and incubated with 40 μL of a plasma sample containing HAMA antibodies with a known VWF activity. The samples used were characterized by the fact that the use of only the HBR-1 reagent could not sufficiently block and thus a falsely high VWF activity was determined.

[0105] Figure 2The results are shown. When the final concentration of the new antibody variant in the final reaction mixture was 0.005 mg / mL, it was already possible to observe an almost complete blockade of the HAMA antibody interference effect. In contrast, the use of only the HBR-1 reagent alone resulted in an insufficient blockade of the HAMA antibody interference effect. The combination of the new antibody variant and the HBR-1 reagent did not show a blockade effect beyond that of the new antibody variant or indicate an impaired blockade effect.

Claims

1. A kit for use in a method for analyzing an analyte in a body fluid sample, the kit comprising: a) A first antigen-specific antibody having a first amino acid sequence that specifically binds an antigen and whose use in a defined test system for analyte detection causes an analyte-specific detection reaction, and b) An antibody variant having a second amino acid sequence and whose antigen-binding ability is significantly reduced compared to that of the first antibody such that its additional use in the defined test system for analyte detection reduces the analyte-specific detection reaction by at most 15%, wherein the amino acid sequence of the antibody variant is identical to that of the first antigen-specific antibody except for one to three modified amino acid residues.

2. The kit according to claim 1, wherein, The 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.

3. The kit according to claim 2, wherein, At least one modified amino acid residue is located in a complementarity-determining region of the heavy chain of the antibody variant.

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.

5. The kit according to any one of the preceding claims, wherein, At least the antibody variant is recombinantly produced.

6. The kit according to any one of the preceding claims, wherein, The first antigen-specific antibody and the antibody variant are present in different reagents.

7. The kit according to any one of the preceding claims, wherein, The first antigen-specific antibody is associated with a solid phase and / or components of a signal-forming system.

8. The kit according to any one of the preceding claims, wherein, The first antigen-specific antibody is an analyte-specific antibody.

9. The kit according to any one of the preceding claims, further comprising: c) At least one additional antigen-specific antibody having a third amino acid sequence that specifically binds an antigen and whose use in a defined test system for analyte detection causes an analyte-specific detection reaction, and d) An additional antibody variant having a fourth amino acid sequence and whose antigen-binding ability is significantly reduced compared to that of the additional antigen-specific antibody such that its additional use in the defined test system for analyte detection reduces the analyte-specific detection reaction by at most 15%, And wherein, wherein the amino acid sequence of the additional antibody variant is identical to that of the additional antigen-specific antibody except for one to three modified amino acid residues.

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 the 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 that of the first GPIb protein-specific antibody except for two modified amino acid residues and has the 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 the preceding claims 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, said interfering antibodies being, for example, from heterophilic antibodies 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, the method comprising the steps of: a) providing a reaction mixture by mixing the sample with i and ii: i. a first antigen-specific antibody having a first amino acid sequence, specifically binding an antigen, and whose use in a defined test system for analyte detection causes an analyte-specific detection reaction, and ii. an antibody variant having a second amino acid sequence and whose antigen-binding ability is significantly reduced compared to that of the first antibody such that its additional use in the defined test system for analyte detection reduces the analyte-specific detection reaction by at most 15%; and b) measuring a measurement variable in the reaction mixture, the measurement variable being affected by the formation of a complex of the antigen with the first antigen-specific antibody and being related to the amount of analyte, wherein the amino acid sequence of the antibody variant is identical to the amino acid sequence of the first antigen-specific antibody, except for one to three modified amino acid residues.

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

18. The method according to any one of claims 16 and 17, wherein The first antigen-specific antibody is an analyte-specific antibody, and wherein the measured measurement variable is affected by the formation of a complex of the analyte with the first analyte-specific antibody.

19. The method according to any one of claims 16 and 17, wherein, The first antigen-specific antibody is an antibody specific for a binding partner of the analyte, and wherein the measured measurement variable is affected by the formation of a complex of the analyte with the binding partner of the analyte and the first antigen-specific antibody specific for the binding partner of the analyte.

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

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

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