Methods and kits for reducing interference 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] This invention relates to an immunoassay in the field of in vitro diagnostic applications and concerns the use of test-specific blocking antibodies to reduce interference caused by heterophilic antibodies or rheumatoid factors. [Background technology]

[0002] Immunoassays have been used for decades in clinical diagnostic tests to quantitatively or qualitatively detect various analytes in bodily fluid samples. Antibodies used for the direct detection of antigens or the indirect detection of different analytes are typically monoclonal or polyclonal animal antibodies obtained from immunized animals (e.g., rabbits, mice, sheep) or by biotechnological means. Misdiagnosis can be widespread due to erroneous test results resulting from interfering substances originating from patient samples. One known and relatively frequent problem is the issue of interfering antibodies that may be endogenously present in an individual's sample (Non-Patent Literature 1). Examples of interfering antibodies include 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 interfering antibodies is so-called rheumatoid factor, i.e., human autoantibodies directed against the Fc portion of human immunoglobulin G. All of these interfering antibodies typically have an affinity for animal antibodies and often bind to the Fc portion. If a testing system uses animal antibodies ("analytical antibodies") as part of a detection reaction to detect an analyte, what may occur in the presence of interfering antibodies from the patient sample is a binding reaction between the analytical antibody and the interfering antibody. This binding reaction may block the detection reaction and result in a false negative / incorrectly low result, or it may enhance the detection reaction and result in a false positive / incorrectly high result.

[0003] Therefore, to reduce such interference, it is standard practice to add blocking antibodies to modern immunoassays. These blocking antibodies are typically a mixture of randomly selected nonspecific antibodies of the same immunoglobulin class and species as the analytical antibody used in the test system. For example, in a test system using monoclonal mouse antibody (e.g., mouse IgG1) as the analytical antibody, an additional mixture of unrelated mouse IgG1, i.e., mouse IgG1 that is non-functional in the test system, may be added. In most cases, this prevents, or at least minimizes, any interfering antibody from the patient sample from binding to the unrelated antibody, thereby preventing problematic binding to the analytical antibody. Such blocking agents are commercially available, for example, Heterophilic Blocking Reagent (HBR) from Scantibodies Laboratory, Inc. or TRU Block reagent from Meridian Bioscience, Inc. The blocking effect of such unrelated antibodies can be optimized by pre-aggregating the unrelated antibodies (Patent Document 1).

[0004] Despite these measures, there are still cases where certain samples cannot be accurately analyzed using specific immunoassay testing systems due to blocking that is clearly ineffective with conventional blocking reagents. Non-patent document 2 describes patient samples in which erroneous high results were repeatedly obtained, despite the addition of HAMA blocking agents (for blocking human anti-mouse antibodies), using two different testing systems to determine von Willebrand factor (VWF) activity, both of which involved the use of monoclonal mouse antibodies in particular.

[0005] In relation to the present invention, “immunoassay” is a method for detecting an analyte in a sample, comprising the use of at least one antigen-specific antibody. The antigen-specific antibody may, but does not necessarily, be an antibody specific to the analyte.

[0006] Depending on the test 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 to directly bind to and detect the analyte; for this purpose, the antibody must be specific to the analyte. In another case, an antibody can be used, for example, to immobilize a binding partner of the analyte to be detected onto a solid phase; for this purpose, the antibody must be specific to the binding partner. Various immunoassay principles are known (direct, indirect, competitive, and non-competitive). What they all have in common is that such principles 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 the detection of an analyte.

[0007] Therefore, a conventional immunoassay optimized to minimize antibody-induced interference is a method for detecting an analyte in a body fluid sample that essentially includes the following steps: i) Sample, a) Antigen-specific antibodies that specifically bind to an antigen, and b) A mixture of nonspecific, randomly selected antibodies that are empirically known to block the binding of interfering antibodies present in the sample to antigen-specific antibodies. By bringing it into contact with, the process of preparing a reaction mixture is completed. ii) A step of measuring a measure 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] US2004 / 0018556A1 [Non-patent literature]

[0009] [Non-Patent Document 1] Bolstad, N. et al., Heterophilic antibody interference in immunometric assays. Best Practice & Research Clinical Endocrinology & Metabolism (2013), pp. 647-661 [Non-Patent Document 2] Bowyer AE et al. (Von Willebrand factor activity assay errors. Haemophilia (2016), 22, pp. e74-e76) [Overview of the project] [Problems that the invention aims to solve]

[0010] Despite the addition of blocking antibodies to the reaction mixture, there are always samples that cannot be accurately analyzed in certain immunoassays due to obviously insufficient blocking. Therefore, one objective 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 factor present in patient samples. [Means for solving the problem]

[0011] The present invention achieves this objective 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 antibody from a patient sample that binds very specifically to the epitope of the analytical antibody and is therefore not blocked or not sufficiently blocked by a randomly selected classical blocking antibody mixture is specifically captured by the induced blocking antibody and consequently blocked. It has been found that antibodies having an amino acid sequence identical to that of the analytical antibody, except for 1 to 3 modified amino acid residues, thereby significantly reducing their antigen-binding ability compared to the analytical antibody, result in efficient blocking of interfering antibodies and thus significantly improve the reliability of immunoassays.

[0012] Therefore, the present invention provides a kit for use in a method for detecting analytes in bodily fluid samples. The kit is i) A first antigen-specific antibody, A first antigen-specific antibody ("analytical antibody") having a first amino acid sequence, specifically binding to the antigen, and whose use results in an analyte-specific detection reaction in a prescribed test system for the detection of the analyte, ii) An antibody variant, Antibody variants (also called "non-analytical antibodies" or "non-analytical antibody variants") have a second amino acid sequence, and their antigen-binding ability is significantly reduced compared to the first antibody, or their competition with the first antigen-specific antibody for binding to the antigen is very low, so that the additional use of antibody variants in a defined test system for analyte detection reduces analyte-specific detection reactions by up to 15%. It contains, 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 explanation of the drawing]

[0013] [Figure 1A]Chart showing the VWF activity [% of norm value] measured in normal sample (1) and sample (2) with reduced VWF activity, with and without the novel antibody variant at various concentrations (mg / mL) in the reaction mixture. [Figure 1B] Chart showing the VWF activity [% of norm value] measured in normal sample (3) and sample (4) with reduced VWF activity, using various dilutions of a commercially available HBR-1 reagent (total protein content in mg / mL of the reaction mixture). [Figure 2] Chart showing the VWF activity [% of norm value] measured in HAMA antibody-containing samples, under the conditions of (1) additional use of various dilutions of a commercially available HBR-1 reagent (total protein content in mg / mL of the reaction mixture), (2) additional use of various concentrations (mg / mL) of the novel antibody variant in the reaction mixture, or (3) additional use of various concentrations (mg / mL) of the novel antibody variant in the reaction mixture combined with the HBR-1 reagent.

Mode for Carrying Out the Invention

[0014] A kit for use in a method for detecting an analyte in a body fluid sample usually 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 and enable a quantitative, semi-quantitative or qualitative determination of the amount or activity of the analyte.

[0015] The first antigen-specific antibody is essential for specifically binding to the antigen and generating the analyte-specific detection reaction desired by the testing system ("analytical antibody"). The antigen-specific antibody may be an analyte-specific antibody that specifically binds to the analyte derived from a body fluid sample. Alternatively, the antigen-specific antibody may specifically bind to the analyte's binding partner. In this case, the analyte's binding partner may be endogenously present in 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 the cleavage product of the analyte.

[0016] Depending on the test 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 to directly bind to and detect the analyte; for this purpose, the antibody must be specific to the analyte. In another case, an antibody can be used, for example, to immobilize a binding partner of the analyte to be detected onto a solid phase; for this purpose, the antibody must be specific to the binding partner. Various immunoassay principles are known (direct, indirect, competitive, and non-competitive). What they all have in common is that such principles 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 the detection of an 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, sheep, camel, or other animal. Preferably, the antibody is a monoclonal antibody or a recombinant 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 complete 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 components of the solid phase and / or signal-forming system.

[0019] The term "solid phase" in relation to the present invention includes articles made of porous and / or non-porous water-insoluble materials, which can take various forms, such as containers, small tubes, microtitration 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 consist of various materials, such as inorganic and / or organic materials, synthetic materials, natural materials, and modified natural materials. Examples of solid phase materials include polymers, such as cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, crosslinked dextran molecules, agarose, polystyrene, polyethylene, polypropylene, polymethacrylate, or nylon; latex; ceramics; glass; metals, especially precious metals such as gold and silver; magnetite; and 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 generate a signal 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 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 aequorin; as sensitizers, eosin, 9,10-dibromoanthracene, methylene blue, porphyrins, phthalocyanins, chlorophyll, rose bengal; coenzymes; as radioisotopes, 3 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 specific interactions with other binding partners, for example, via specific interactions 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 derived from a first antigen-specific antibody or a corresponding antibody fragment (also called a "non-analytical antibody" or "non-analytical antibody variant"), and has: 1) an amino acid sequence identical to the amino acid sequence of the first antigen-specific antibody except for 1 to 3 modified amino acid residues; and 2) antigen-binding ability which is significantly reduced compared to the first antigen-specific antibody, resulting in an analyte-specific detection reaction being reduced by up to 15% by the additional use of the antibody variant in a specified test system for analyte detection.

[0024] Therefore, such “non-analytical” antibody variants are typically available by individually tailoring them to a first antigen-specific antibody (“analytical antibody”) and by defining 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 1 to 3 amino acid residues, and then recombinantly generating the modified antibody variant accordingly. The positions of one, two, or three modified amino acid residues should be selected to be in the region of the analytical antibody such that they are related to antigen binding and will be functionally restricted or inactivated by the modification of the amino acid sequence, and the antigen-binding ability of the resulting modified non-analytical antibody variant is lacking or at least significantly reduced. For this purpose, it is preferable that 1 to 3 amino acid residues are modified, i.e., substituted, deleted, inserted, or chemically derivatized, in one or more complementarity-determining regions (CDRs) of the heavy or light chain of the analytical antibody. The complementarity-determining regions (CDRs) (CDR-H1, CDR-H2, and CDR-H3) of the antibody heavy chain and the complementarity-determining regions (CDRs) (CDR-L1, CDR-L2, and CDR-L3) (according to the Kabat numbering scheme) that are separated from each other by so-called framework regions are well known to those skilled in the art. Particularly preferably, at least one amino acid residue is modified within the complementarity-determining region of the analytical antibody heavy chain. More preferably, at least one amino acid residue is modified within the CDR-H3 complementarity-determining region of the analytical antibody heavy chain.

[0025] Therefore, in one embodiment of the test kit, an antibody variant exists in which 1 to 3 modified amino acid residues are located in one or more complementarity-determining regions (CDRs) of the heavy or light chain of the antibody variant.

[0026] In a further embodiment of the test kit, an antibody variant exists 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 test kit, there exists an antibody variant in which at least one modified amino acid residue is located in the complementarity-determining region CDR-H3 of the antibody variant's heavy chain.

[0028] "Modified amino acid residues" should be understood as amino acid residues that have been substituted, deleted, inserted, or chemically derivatized in relation to the consecutive positions of amino acids in the primary sequence of an 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 include amino acids with basic side chains, amino acids with acidic side chains, amino acids with nonpolar aliphatic side chains, amino acids with nonpolar aromatic side chains, amino acids with polar side chains, amino acids with uncharged 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 may be replaced by a large amino acid residue, or a charged amino acid residue may be replaced by an uncharged amino acid residue.

[0029] "Non-analytical" antibody variants are also naturally occurring variants of the primary 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] Therefore, the antibody variant is either a non-functional variant of the first antigen-specific antibody or at least a less functional variant of it in terms of its antigen-binding ability.

[0031] The antigen-binding ability of the antibody variant must be significantly reduced compared to the primary antigen-specific antibody, resulting in a reduction of up to 15% in analyte-specific detection reactions through the additional use of the antibody variant in a defined test system for analyte detection.

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

[0033] However, it is crucial that the antigen-binding ability of an antibody variant be tested in the same defined testing system for detecting the analyte, where the first antigen-specific antibody is used as the “analytical” antibody. The term “defined testing system” refers to a defined testing setup with respect to the components and method steps used. A variation of a single component or method step in a testing setup that is otherwise unchanged makes it possible to determine the effect of the variation in a otherwise defined testing system. Ideally, the method for detecting the analyte is used as the defined testing system, in which case the use of the first antigen-specific antibody and the antibody variant is intended to achieve this. In relation to the present invention, “used for this purpose” refers to 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 a testing system where the primary antigen-specific antibody is used as the “analytical” antibody, appropriate antibody variants do not have a significant competitive effect on the analyte-specific detection reaction. This is ensured by first measuring the reaction intensity of the analyte-specific detection reaction across the entire measurement range in the testing system, both with and without the addition of the antibody variant to the reaction mixture in the sample without interfering antibodies. A suitable antibody variant is one that has significantly reduced antigen-binding capacity compared to the first antigen-specific antibody, and as a result, its presence does not reduce the reaction intensity of the analyte-specific detection reaction by more than 15%, preferably more than 10%, and particularly preferably more than 5%; the reduction in the antigen-binding capacity of the antibody variant is functionally demonstrated by: little to no competition between the antibody variant and the first antigen-specific antibody for binding to the antigen, i.e., little to no competition for the antigen binding site, and as a result, the use of the antibody variant in combination with the first antigen-specific antibody does not have an excessively problematic effect on the analyte-specific detection reaction, but instead 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 present invention, the first antigen-specific antibody and 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, at the bottom of a well in a microtiter plate, or inside a reaction tube. Such a kit is particularly suitable for performing heterogeneous diagnostic methods, such as ELISA. Such a kit preferably further includes a further container containing the antibody variant as a component of a liquid reagent (or its lyophilized form).

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

[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 the component of the signal-forming system, or it 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 includes a container containing the corresponding reagent in liquid form or as a resuspendable lyophilized version thereof. Depending on the nature of the signal-forming system, such a test kit is suitable for measuring, for example, chemiluminescence, fluorescence, or absorption changes.

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

[0040] 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 used in a sandwich immunoassay, in which case the second antigen-specific antibody may be specific to the same antigen as the first antigen-specific antibody, or it may be specific to 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 the second antigen-specific antibody with respect to its antigen-binding ability, or at least a less functional variant of it. Therefore, such kits are c) A second antigen-specific antibody, A second antigen-specific antibody having a third amino acid sequence, specifically binding to the antigen, and whose use results in an analyte-specific detection reaction in a prescribed test system for analyte detection, d) Further antibody variants, The fourth amino acid sequence is present, and the antigen-binding ability of this further antibody variant is significantly reduced compared to the second antigen-specific antibody. As a result, the additional use of this further antibody variant in a defined test system for analyte detection reduces the analyte-specific detection reaction by up to 15%. It also contains, The amino acid sequences of the further antibody variants are identical to those of the second antigen-specific antibody, with the exception of 1 to 3 modified amino acid residues.

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

[0042] The present invention further provides the use of a kit according to the present invention in a method for detecting analytes in bodily fluid samples.

[0043] Particularly preferable is the use of the kit according to the present invention for uninterfering detection of analytes in bodily fluid samples containing interfering antibodies derived from groups such as heterophilic antibodies and autoantibodies.

[0044] In a specific embodiment of the kit, the first antigen-specific antibody is an antibody that specifically binds to glycoprotein Ib (GPIb) protein. GPIb protein is a binding partner of von Willebrand factor (VWF) and is used in various assays for determining VWF activity (see, e.g., 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 by configuring a test method such that the formation of a complex between VWF and GPIb in the test reaction can be measured by measuring the aggregation of latex particles, for example, latex particles coated with an anti-GPIb antibody, which aggregate only when a VWF-GPIb complex is formed in the test reaction and subsequently bound by the anti-GPIb antibody associated with the latex particles. As mentioned above, in such assays using mouse monoclonal anti-GPIb antibodies, it has been observed that falsely high results are repeatedly obtained despite the addition of HAMA blockers (for blocking human anti-mouse antibodies).

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

[0046] Such test kits are suitable for use in methods for detecting VWF activity in bodily fluid samples and have the special advantage of enabling uninterrupted detection of VWF activity in bodily fluid samples containing interfering antibodies derived from heterophilic antibodies and autoantibodies, for example.

[0047] The present invention further provides a method for detecting analytes in bodily fluid samples, the method being: a) Sample i. A first antigen-specific antibody, It has a first amino acid sequence, binds specifically to the antigen, and its use results in an analyte-specific detection reaction in a prescribed test system for analyte detection. The first antigen-specific antibody, and ii. Antibody variant, Having a second amino acid sequence, the antigen-binding ability of this antibody variant is significantly reduced compared to the first antibody. As a result, the additional use of this antibody variant in a specified test system for analyte detection reduces analyte-specific detection reactions by up to 15%. Antibody variant The process involves preparing a reaction mixture by mixing it with the following: b) A step of measuring a measure 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. Includes, Here again (as already mentioned above), 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.

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

[0049] In one embodiment of the method according to the present invention, the first antigen-specific antibody is an analyte-specific antibody, and the measured variable is affected by the formation of a complex between the analyte and the first analyte-specific antibody. One example is an immunoassay, in which a complex is formed between the analyte and a latex particle-associated analyte-specific antibody, and the formation of this complex is determined by photometry based on the agglutination reaction of latex particles in the reaction mixture.

[0050] In another embodiment of the method according to the present invention, the first antigen-specific antibody is an antibody specific to the binding partner of the analyte, and the measured variable is influenced by the formation of a complex between the analyte, the binding partner of the analyte, and the first antigen-specific antibody specific to the binding partner of the analyte. One example is a functional binding test, which measures not the amount of the analyte but rather the ability of the analyte to bind to a specific binding partner, in which case a complex is formed between the analyte, the binding partner of the analyte, and, for example, a latex particle association antibody specific to the binding partner, and the formation of the complex is determined by photometry based on the agglutination reaction of latex particles in the reaction mixture.

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

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

[0053] The aggregation of particulate solid phases in the reaction mixture can be measured by photometric methods, such as turbidity or turbidimetry. Coupling tests based on the principle of particle-enhanced light scattering have been known since around 1920 (for a review, see Newman, DJ et al., Particle enhanced light scattering immunoassay. Ann Clin Biochem 1992;29:22-42). In this regard, polystyrene particles having a diameter of 0.1-0.5 μm are preferred, and polystyrene particles having a diameter of 0.15-0.35 μm are more preferred. It is preferable to use polystyrene particles having amine, carboxyl, or aldehyde functional groups. It is also preferable to use core-shell type particles. For particle synthesis and ligand covalent bonding, see, for example, Peula, JM 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, the aggregation of 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 spatial proximity. In this context, a first fraction of the particulate solid phase associates with a first component of the signal-forming system, and a second fraction of the particulate solid phase associates with a second component of the signal-forming system. The first and second components of the signal-forming system cooperate to generate a detectable signal when they are in spatial proximity, and the aggregation of particulate solid phase in the reaction mixture is measured based on the generated signal.

[0055] In this embodiment of the method according to the present invention, the signal-forming system comprises at least first and second components which cooperate to generate a detectable signal when they are in spatial proximity and thereby able to interact with each other. The interaction between the two components is understood to mean energy transfer in particular, i.e., direct transfer of energy between the two components, for example, through irradiation with light or electrons or via reactive chemical molecules such as short-lived singlet oxygen. The energy transfer may be from one component to the other, or a cascade of various materials through which energy transfer proceeds is also conceivable. For example, both components can be pairs comprising an energy donor and an energy acceptor, such as a photosensitizer and a chemiluminescent agent (EP-A2-0515194, LOCI® Technology) or a photosensitizer and a phosphor (WO95 / 06877) or radioactive iodine-125 and a phosphor (Udenfriend et al. (1985) Proc. Natl. Acad. Sci. 82: pp. 8672-8676) or a phosphor and a fluorescent quencher (US3,996,345). Particularly preferred is that 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 what is measured is the chemiluminescence in the reaction mixture.

[0056] The following examples and figures are illustrative of the present invention and should not be understood as limiting it. [Examples]

[0057] Example 1: Latex aggregation assay for determining VWF activity using conventional technology Reagent 1: HBR-1 reagent (Heterophilic Blocking Reagent 1, Scantibodies Laboratory, Inc., Santee, USA) contains a mixture of mouse immunoglobulins for binding to heterophilic antibodies.

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

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

[0060] The von Willebrand factor (VWF) activity in plasma samples was determined as follows: 1. Mix 40 μL of sample with 12 μL of reagent, and incubate the prepared sample at room temperature for 30 minutes. 2. Next, 15 μL of the pre-treated sample was mixed with 30 μL of Owren's Veronal buffer, with 70 μL of buffer containing an additional surfactant, and with 15 μL of reagent 2, and the mixture was incubated at 37°C for 2 minutes. 3. Next, 40 μL of reagent 3 was added to the mixture, and the change in absorbance of the reaction mixture was measured using light at a wavelength of 570 nm. 4. The measured raw values ​​were evaluated using a calibration curve.

[0061] Despite the use of the HBR-1 reagent, there are occasionally samples that yield falsely high results due to the clearly ineffective blocking of HAMA intended by reagent 1.

[0062] Example 2: Generation of an anti-GPIb antibody variant with 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] Complementarity Determination Region [ka] The amino acid sequence (according to the Kabat numbering scheme) in the variable chain region of the heavy chain of the anti-GPIb antibody, including the following, was as follows: [ka]

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

[0065] Therefore, the complementarity determination domain [ka] The amino acid sequence in the variable chain region of the heavy chain of the modified antibody, including the following, was as follows: [ka]

[0066] Example 3: Detection of reduced GPIb binding ability of novel antibody variants The testing system used was a latex agglutination assay for determining VWF activity, as described in Example 1.

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

[0068] The results are shown in Figure 1A. For the novel antibody variant in the final reaction mixture, the measured VWF activity was reduced by up to 2.3% at final concentrations down to 0.02 mg / mL compared to the reaction mixture without 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 its binding site on the GPIb protein.

[0069] The novel 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. However, it exhibits a significantly reduced GPIb binding capacity compared to the functional anti-GPIb antibody.

[0070] For comparison, the assay was modified in terms of further variants, by mixing 2 μL of various dilutions of Reagent 1 (HBR-1 Reagent) containing various total protein concentrations with 40 μL of normal plasma sample or 40 μL of a sample known to have reduced VWF activity, and incubating them in each case.

[0071] The results are shown in Figure 1B. At final concentrations of total HBR protein in the final reaction mixture down to 0.02 mg / mL, the measured VWF activity was reduced by up to 2.6% compared to the reaction mixture without 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 due to a specific antibody variant.

[0072] Example 4: Detection of HAMA antibody blocking effect of novel antibody variants The latex agglutination assay for determining VWF activity according to Example 1 was modified by mixing and incubating 2 μL of reagent containing either a different amount of the novel antibody variant generated according to Example 2 alone, or a different amount of the novel antibody variant generated according to Example 2 in combination with Reagent 1 (HBR-1 reagent), with 40 μL of a plasma sample containing a HAMA antibody with known VWF activity. The samples used were identified by the fact that they could not be adequately blocked by the use of HBR-1 reagent alone, resulting in the determination of falsely high VWF activity.

[0073] The results are shown in Figure 2. At final concentrations starting from 0.005 mg / mL, the novel antibody variant in the final reaction mixture exhibits nearly complete blocking of the HAMA antibody interference effect. In contrast, the use of HBR-1 reagent alone results in only insufficient blocking of the HAMA antibody interference effect. The combination of the novel antibody variant with HBR-1 reagent did not show a blocking effect exceeding that of the novel antibody variant alone, nor a blocking effect indicating impaired blocking.

Claims

1. A kit for use in a method for detecting analytes in bodily fluid samples, a) A first antigen-specific antibody, It has a first amino acid sequence, Binds specifically to the antigen, Its use results in a first antigen-specific antibody that produces an analyte-specific detection reaction in a prescribed testing system for detecting the analyte, b) An antibody variant, It has a second amino acid sequence, The antigen-binding ability of this antibody variant is significantly reduced compared to the first antigen-specific antibody, and as a result, the additional use of the antibody variant in a specified test system for analyte detection reduces the analyte-specific detection reaction by up to 15%. It contains, The amino acid sequence of the antibody variant is characterized by being identical to that of the first antigen-specific antibody, except for 1 to 3 modified amino acid residues. kit.

2. The kit according to claim 1, wherein one to three modified amino acid residues are located in one or more complementarity-determining regions (CDRs) of the heavy chain 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 one 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 claims 1 to 4, wherein at least the antibody variant is generated by recombinant means.

6. The kit according to any one of claims 1 to 5, wherein the first antigen-specific antibody and antibody variant are present in different reagents.

7. The kit according to any one of claims 1 to 6, wherein the first antigen-specific antibody is associated with a component of the solid phase and / or a signal-forming system.

8. The kit according to any one of claims 1 to 7, wherein the first antigen-specific antibody is a specific antibody of the analyte.

9. c) At least one further antigen-specific antibody, It has a third amino acid sequence, Binds specifically to the antigen, Its use results in an analyte-specific detection reaction in a defined testing system for detecting the analyte. At least one further antigen-specific antibody, d) Further antibody variants, It has a fourth amino acid sequence, The antigen-binding ability of these additional antibody variants is significantly reduced compared to additional antigen-specific antibodies, and as a result, the additional use of these additional antibody variants in a defined test system for analyte detection can reduce analyte-specific detection reactions by up to 15%. It further contains, The amino acid sequences of the further antibody variants are identical to those of the further antigen-specific antibodies, except for one to three modified amino acid residues. A 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 heavy chain complementarity-determining region CDR-H3.

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 the heavy chain complementarity determination region CDR-H3 has an amino acid sequence according to SEQ ID NO:

2.

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

14. Use of the kit according to any one of claims 1 to 12 for detecting an analyte without interference in a body fluid sample containing interfering antibodies derived from groups of 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 analytes in a body fluid sample, a) Sample i. A first antigen-specific antibody, Having the first amino acid sequence, Binds specifically to the antigen, Its use results in an analyte-specific detection reaction in a prescribed testing system for detecting the analyte. The first antigen-specific antibody, and ii. Antibody variant, It has a second amino acid sequence, The antigen-binding ability of this antibody variant is significantly reduced compared to the first antigen-specific antibody. As a result, the additional use of the antibody variant in a specified test system for analyte detection can reduce analyte-specific detection reactions by up to 15%. Antibody variant The process involves preparing a reaction mixture by mixing it with the following: b) A step of measuring a measure 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. Includes, The amino acid sequence of the antibody variant is characterized by being identical to that of the first antigen-specific antibody, except for 1 to 3 modified amino acid residues. method.

17. The method according to claim 16, comprising first mixing a sample with an antibody variant, incubating the mixture thus produced, and then adding one antigen-specific antibody to the mixture.

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

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

20. The first antigen-specific antibody is an antibody that has specificity for the GPIb protein. The measured parameters are influenced by the formation of a complex between von Willebrand factor, GPIb protein, and a first antigen-specific antibody that has specificity for GPIb protein. The method according to claim 19 for detecting the activity of von Willebrand factor in a bodily fluid sample.

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

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