Immunological assay method, immunological assay reagent, immunological assay sample pretreatment liquid, immunological assay kit, and non-specific reaction inhibitor

Abstract

An immunological assay method for measuring a target substance in a sample, wherein at least one antigen-antibody reaction is performed in the presence of an enzyme that specifically decomposes immunoglobulin A (IgA).

Description

Technical Field

[0001] This invention relates to immunological assay methods, reagents for immunological assays, sample pretreatment solutions for immunological assays, kits for immunological assays, and nonspecific reaction inhibitors. This application claims priority based on Provisional Application 63 / 559,187, filed February 29, 2024, the contents of which are incorporated herein by reference. Background Technology

[0002] As a diagnostic reagent, one example of an immunological assay is the use of antigen-antibody reactions to detect the analyte in biological samples. Immunological assays utilize antigen-antibody reactions, making them highly specific assays.

[0003] Various substances exist in biological samples. Non-specific binding reactions can occur due to substances other than the target substance, or specific antigen-antibody reactions can be hindered, resulting in measurement errors. Such phenomena are called non-specific reactions, and substances that cause non-specific reactions are called non-specific factors, etc.

[0004] As non-specific factors, the presence of heterophile antibodies and rheumatoid factor (RF) has been confirmed. Heterophile antibodies are a collective term for human antibodies that exhibit reactivity to animal-derived antibodies, which are responsible for the main reaction in immunological assays; human anti-mouse immunoglobulin antibody (HAMA) is a representative example. Rheumatoid factor is a glycoprotein identified at a high rate in patients with collagen diseases such as rheumatoid arthritis, chronic infections, and liver diseases, and its reactivity to animal-derived antibodies is similar to that of HAMA. RF is typically detected with IgM-RF being the most common type of IgM, but the presence of IgA-RF has also been confirmed (Non-Patent Literature 1, 3).

[0005] Furthermore, immunoglobulin A (hereinafter referred to as IgA) is known to cause nonspecific reactions in immunological assays (Non-Patent Document 2).

[0006] As a technique for suppressing nonspecific reactions in immunological assays, patent documents 1, 2 and 3 are known, for example.

[0007] Patent Document 1 discloses a method for suppressing nonspecific reactions caused by rheumatoid factor (RF) by pretreating a sample with a sufficient amount of animal-derived antibody having the ability to bind to the antigen-binding site (Fab) of human rheumatoid factor. Examples of such animal-derived antibodies include anti-human immunoglobulin Fab antibodies, anti-human IgG antibodies (Fab-specific), anti-human IgA antibodies (Fab-specific), and anti-human IgM antibodies (Fab-specific).

[0008] Patent document 2 discloses a method for suppressing non-specific reactions caused by interfering substances having a structure in which polypeptide chains, such as rheumatoid factor, are linked together by disulfide bonds. This is achieved by using a reducing agent to break the disulfide bonds and decompose the interfering substances.

[0009] Patent document 3 discloses a method in which a liquid sample containing the protein of the test subject and one or more other proteins other than the test subject are mixed with a protein digesting agent, and protein digestion is performed under the condition that the antigenicity determination factor of the protein of the test subject is provided to the mixture, thereby reducing or eliminating interference caused by proteins other than the test subject.

[0010] Existing technical documents

[0011] Patent documents

[0012] Patent Document 1: Japanese Patent Application Publication No. 07-012818

[0013] Patent Document 2: Japanese Patent Application Publication No. 13-255325

[0014] Patent Document 3: Japanese Patent Publication No. 02-021548

[0015] Non-patent literature

[0016] Non-patent literature 1: Clinical Chemistry; Volume 23 Supplement 175a-1~175a-10 (1994)

[0017] Non-Patent Literature 2: Tokushima Red Cross Hospital Medical Journal; Volume 18, No. 1, pp. 56-60 (2013)

[0018] Non-patent literature 3: Immunotestology (Immunotestology). Tetsuro Kubota, Kiyotaka Fujita, Eiji Hosoi, Michiko Kajiwara (Tetsuro Kubota, Kiyotaka Fujita, Eiji Hosoi, Michiko Kajiwara). Medical and Dental Publishing Co., Ltd. P.199 (2017) Summary of the Invention

[0019] The technical problem that the invention aims to solve

[0020] The anti-human IgM polyclonal antibody, anti-human IgG polyclonal antibody, and anti-human IgA polyclonal antibody shown in Patent Document 1 are currently being used in various reagents to inhibit non-specific reactions caused by natural antibodies and RF, but it has been clarified that this method cannot adequately inhibit non-specific reactions.

[0021] The reducing agent shown in Patent Document 2, used to suppress non-specific reactions caused by interfering substances with structures in which polypeptide chains are linked together by disulfide bonds, may cleave the disulfide bonds of the antigen or antibody used as the analyte, or the antigen or antibody contained in the reagent components, depending on the type and concentration, resulting in low versatility. Furthermore, in methods using reducing agents, molecules with disulfide bonds, such as IgG and IgA, may not be distinguished and may be decomposed. Therefore, this method is particularly unusable when human IgG is used as the analyte.

[0022] Examples of protein digestive agents shown in Patent Document 3 include pepsin, papain, and trypsin. Pepsin exhibits optimal enzyme activity in the acidic pH range of 1-3, therefore the analyte and / or substances specifically bound to it may be modified or decomposed. Papain and trypsin exhibit enzyme activity on a wide range of substances, therefore the analyte and / or substances specifically bound to it may be modified or decomposed.

[0023] The purpose of this invention is to provide an immunological assay method that can suppress non-specific reactions caused by IgA contained in the assay sample.

[0024] Technical solutions for solving technical problems

[0025] To address the aforementioned issues, the inventors investigated the inhibitory effects of various substances on non-specific reactions. They discovered that by conducting antigen-antibody reactions in the presence of an enzyme that specifically breaks down IgA, non-specific reactions could be inhibited, thus completing this invention. Specifically, this invention has the following structure.

[0026] [1] An immunological assay method, wherein in the immunological assay method for measuring the analyte in a sample, at least one antigen-antibody reaction is performed in the presence of an enzyme that specifically breaks down immunoglobulin A (IgA).

[0027] [2] According to the immunological assay method described in [1], wherein the enzyme is a protease.

[0028] [3] According to the immunological assay method described in [2], the protease is an endopeptidase or an exopeptidase.

[0029] [4] An immunological assay method according to any one of [1] to [3], wherein the enzyme is an enzyme that cleaves an amino acid sequence containing the IgA constant region from CHα1 to CHα3.

[0030] [5] An immunological assay method according to any one of [1] to [4], wherein the enzyme is an enzyme that acts on IgA in the sample to decompose the IgA into Fab fragments and Fc fragments.

[0031] [6] The immunological assay method according to any one of [1] to [5], wherein the IgA is IgA1.

[0032] [7] The immunological assay method according to any one of [1] to [6], wherein the immunological assay method is latex immunoturbidimetric assay.

[0033] [8] An immunological assay method according to any one of [1] to [7], wherein the assay object is an antigen or an antibody.

[0034] [9] An immunological assay reagent for use in an immunological assay method for determining a analyte in a sample, the reagent comprising an enzyme that specifically breaks down IgA.

[0035]

[10] The immunoassay reagent according to [9], wherein the enzyme is a protease.

[0036]

[11] The immunoassay reagent according to

[10] , wherein the protease is an endopeptidase or an exopeptidase.

[0037]

[12] The immunoassay reagent according to any one of [9] to

[11] , wherein the enzyme is an enzyme that cleaves an amino acid sequence containing the IgA constant region from CHα1 to CHα3.

[0038]

[13] The immunoassay reagent according to any one of [9] to

[12] , wherein the enzyme is an enzyme that acts on IgA in the sample to decompose the IgA into Fab fragments and Fc fragments.

[0039]

[14] The immunoassay reagent according to any one of [9] to

[13] , wherein the IgA is IgA1.

[0040]

[15] The immunoassay reagent according to any one of [9] to

[14] , wherein the immunoassay reagent is a reagent for latex immunoturbidimetric assay.

[0041]

[16] The immunoassay reagent according to any one of [9] to

[15] , wherein, in the immunoassay method, the analyte is an antigen or antibody.

[0042]

[17] A sample pretreatment solution for an immunoassay, comprising an enzyme that specifically breaks down IgA.

[0043]

[18] The sample pretreatment solution for immunoassay according to

[17] , wherein the enzyme is a protease.

[0044]

[19] The sample pretreatment solution for immunoassay according to

[18] , wherein the protease is an endopeptidase or an exopeptidase.

[0045]

[20] The sample pretreatment solution for immunoassay according to any one of

[17] to

[19] , wherein the enzyme is an enzyme that cleaves the amino acid sequence containing the IgA constant region from CHα1 to CHα3.

[0046]

[21] The sample pretreatment solution for immunoassay according to any one of

[17] to

[20] , wherein the enzyme is an enzyme that acts on IgA in the sample to decompose the IgA into Fab fragments and Fc fragments.

[0047]

[22] The sample pretreatment solution for immunological assay according to any one of

[17] to

[21] , wherein the IgA is IgA1.

[0048]

[23] The sample pretreatment solution for immunoassay according to any one of

[17] to

[22] , wherein the sample pretreatment solution for immunoassay is a solution for latex immunoturbidimetric assay.

[0049]

[24] An immunoassay kit for an immunoassay method for determining an analyte in a sample, the kit comprising an enzyme that specifically breaks down IgA.

[0050]

[25] The immunoassay kit according to

[24] , wherein the enzyme is a protease.

[0051]

[26] According to the immunoassay kit of

[25] , wherein the protease is an endopeptidase or an exopeptidase.

[0052]

[27] An immunoassay kit according to any one of

[24] to

[26] , wherein the enzyme is an enzyme that cleaves an amino acid sequence containing the IgA constant region from CHα1 to CHα3.

[0053]

[28] An immunoassay kit according to any one of

[24] to

[27] , wherein the enzyme is an enzyme that acts on IgA in the sample to decompose the IgA into Fab fragments and Fc fragments.

[0054]

[29] An immunoassay kit according to any one of

[24] to

[28] , wherein the IgA is IgA1.

[0055]

[30] A nonspecific reaction inhibitor comprising an enzyme that specifically breaks down IgA.

[0056] Invention Effects

[0057] According to the present invention, an immunological assay method is provided that can suppress nonspecific reactions caused by IgA contained in the assay sample.

[0058] In the immunological assay method of the present invention, non-specific reactions that have previously failed to be suppressed even when using non-specific reaction inhibitors can be suppressed. As a result, accurate determination of the analyte can be performed. Detailed Implementation

[0059] [Immunological Assay Methods]

[0060] The immunological assay method of the present invention is characterized in that, in the method for immunologically measuring the analyte in a sample, at least one antigen-antibody reaction is performed in the presence of an enzyme that specifically decomposes IgA. In other words, the method is as follows: an enzyme that specifically decomposes IgA is reacted with the sample as a non-specific reaction inhibitor, and in the presence of this enzyme, an immunological assay of the analyte in the sample is performed using a specific binding coupler.

[0061] One aspect of the immunological assay method of the present invention is a non-specific reaction inhibition method, in which at least one antigen-antibody reaction is performed in the presence of an enzyme that specifically breaks down IgA, thereby inhibiting the non-specific reaction in the reaction solution.

[0062] Immunological assays are broadly classified into homogeneous assays and heterogeneous assays.

[0063] Homogeneous methods are assays that specifically detect the binding reaction between the analyte and its specific binding pair in a mixed solution (reaction solution) of the sample and reagent without B / F (bound / unbound) separation. Heterogeneous methods, on the other hand, involve B / F separation, washing, and removal of unreacted components before proceeding with the binding reaction to detect the analyte.

[0064] Heterogeneous methods, due to the cleaning process, involve multiple steps and require time for measurement. However, they are less susceptible to the influence of non-specific reactants. In contrast, homogeneous methods, which do not involve a cleaning process, are more susceptible to non-specific reactions. However, they are simpler and require fewer steps, resulting in shorter measurement times. Therefore, they are widely sought after in the field of clinical diagnostics.

[0065] As a homogeneous method, examples include assays utilizing immunoagglutination (IA), such as immunoturbidimetry (TIA) and immunochromatography (side-flow and cross-flow). TIA is a method for qualitatively or quantitatively detecting the analyte in a sample by utilizing the degree of aggregation of an immune complex formed through the cross-linking of the analyte with antibodies or other specific binding agents. Among these, latex immunoturbidimetry (hereinafter sometimes called LTIA), which uses latex particles as an insoluble carrier to amplify the aggregation signal, is a highly versatile assay suitable for optical detection, easy to automate, and therefore applicable to various examinations.

[0066] Examples of heterogeneous methods include ELISA, which uses trap plates, and chemiluminescence methods.

[0067] The present invention can be used in any of the above-mentioned immunological assays, but the homogeneous method, which is more susceptible to non-specific reactions, is expected to have further benefits. Therefore, the most preferred method among the homogeneous methods is latex immunoturbidimetry.

[0068] [Enzymes that specifically break down IgA]

[0069] As one aspect of the present invention, examples include methods for immunological assays using enzymes that specifically break down IgA, or non-specific reaction inhibitors containing such enzymes (hereinafter, enzymes that specifically break down IgA are sometimes abbreviated as IgA-specific degrading enzymes). IgA-specific degrading enzymes are any enzymes capable of specifically breaking down IgA; for example, proteases are examples. Among proteases, examples include endopeptidases, exopeptidases, metalloproteinases, serine proteases, cysteine ​​proteases, etc. Furthermore, in the present invention, the term "IgA-degrading enzyme" simply refers to an enzyme that cleaves the amino acid sequence of the constant region of IgA containing CHα1 to CHα3; preferably, it is an enzyme that cleaves a portion of the amino acid sequence in the hinge region between CHα1 and CHα2 (e.g., VPSTPPTPSPST: sequence number 1 of human IgA); more preferably, it is an enzyme capable of breaking down IgA into Fab and Fc fragments. It should be noted that an IgA-specific degrading enzyme only needs to have the function of specifically degrading IgA when in contact with samples or other test materials. The enzyme can be inactivated during the subsequent antigen-antibody reaction.

[0070] In this specification, the terms "antibody reacts" with antigen and "antibody recognizes" antigen are used interchangeably but are not limited to these examples and should be interpreted in the broadest sense. Confirmation of whether an antibody "reacts" with an antigen can be performed using methods such as antigen-immobilized ELISA, competitive ELISA, and sandwich ELISA, as well as methods utilizing the surface plasmon resonance principle (SPR). The SPR method can be performed using devices, sensors, and reagents commercially available under the name Biacore (registered trademark).

[0071] In this specification, the terms "enzyme breaks down" IgA, "enzyme reacts with IgA", and "enzyme digests" IgA are used with the same meaning, but are not limited to these examples. The meaning of "enzyme breaks down" IgA should be interpreted in the broadest sense.

[0072] The IgA-specific degrading enzyme of this invention specifically degrades IgA. Compared with immunoglobulins other than IgA, such as IgG and IgM, the IgA-specific degrading enzyme can efficiently degrade IgA. Therefore, whether the analyte is IgG or the binding partner that specifically binds to the analyte is IgG, the IgA-specific degrading enzyme can be used in a manner that does not affect the specificity of the analyte. Furthermore, the IgA-specific degrading enzyme can degrade IgA derived from organisms such as humans, mice, rats, rabbits, goats, ostriches, and pigs. When it is desired to specifically degrade human IgA, a human IgA-specific degrading enzyme is preferred. In particular, human IgA contains IgA1 and IgA2 as subclasses. The human IgA-specific degrading enzyme can specifically degrade human IgA1 and human IgA2, and more preferably specifically degrades human IgA1. Additionally, IgA exists in blood and secretions, mainly as monomers in blood, and as dimers and polymers in secretions. IgA-specific degrading enzymes preferably break down both IgA present in the blood and IgA present in secretions, but they can also break down only one of them.

[0073] In this invention, the so-called IgA-specific degrading enzyme specifically degrades IgA refers to, for example, the effect of reacting IgG or IgA with the enzyme at 37°C for 30 to 60 minutes, such that the amount of residual IgA is 50% or less, 40% or less, 30% or less, preferably 20% or less, and more preferably 10% or less compared to IgG.

[0074] The IgA-specific degrading enzyme used in this invention can be a naturally occurring enzyme or a recombinant enzyme produced through gene recombination technology. Examples of naturally occurring enzymes include enzymes produced by various bacteria, fungi, etc., such as enzymes produced by bacteria that cause oral infections (Streptococcus sanguis, Streptococcus mitis, and Streptococcus oralis), bacteria that cause genital tract infections (Neisseria gonorrhoeae), and bacteria that cause meningitis (Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae). (Patricia de Sousa-Pereira and Jenny M. Woof. (2019). IgA: Structure, Function, and Developability. Antibodies, 8, 57.) IgASAP (manufactured by Genovis) or amino acid mutants of this enzyme are preferred examples. Alternatively, recombinant enzymes produced as recombinant proteins can also be used. By artificially introducing amino acid residue substitutions into the recombinant enzyme and screening using various known analytical systems, recombinant enzyme variants with desired enzyme activity can be produced (Japanese Patent Application Laid-Open No. 2019-506866, EP3148576). Therefore, in this invention, non-specific reaction inhibition methods in immunological assays can be implemented using various IgA-specific degrading enzymes, or non-specific reaction inhibitors can be produced.

[0075] In this invention, as a method for presenting an enzyme that specifically degrades IgA within an antigen-antibody reaction system, examples include methods for using it as a reagent component in an immunoassay reagent, and methods for adding it to sample diluents, sample extracts, sample pretreatment solutions, etc. For example, sample diluents, sample extracts, or sample pretreatment solutions pre-containing an IgA-specific degrading enzyme can be used, or the IgA-specific degrading enzyme can be added directly to the sample diluent, sample extract, or sample pretreatment solution. It should be noted that in this specification, the term "sample pretreatment solution" includes both sample diluents and sample extracts.

[0076] For example, when the reagents used in immunological assays are liquid reagents, the antigen-antibody reaction system refers to the liquid phase in which the sample is mixed with the liquid immunological assay reagents and the antigen-antibody reaction takes place.

[0077] For example, in the case of LTIA, the sample can be mixed with an immunoassay reagent containing an IgA-specific degrading enzyme, or the sample can be pre-mixed with a sample pretreatment solution containing an IgA-specific degrading enzyme before being mixed with the LTIA reagent.

[0078] In the case of ELISA, the sample can be pre-mixed with IgA-specific degrading enzyme and then dropped into the microplate, or the sample can be mixed with a solution containing IgA-specific degrading enzyme and detection antibody and then dropped into the microplate.

[0079] In the case of chemiluminescence, the sample can be pre-mixed with a sample pretreatment solution containing an IgA-specific degrading enzyme and then mixed with the immunoassay reagent. Alternatively, the immunoassay reagent (e.g., a solution containing a detection antibody or antigen, or magnetic particles) can contain the IgA-specific degrading enzyme.

[0080] In solid-phase antigen-antibody reactions, such as those performed by immunochromatography, the antigen-antibody reaction system refers to the solid phase in which the liquid sample reacts with the binding partner antibody. In this case, the sample can be pre-mixed with a sample pretreatment solution containing an IgA-specific degrading enzyme and then added dropwise to an immunochromatographic test strip. Alternatively, the IgA-specific degrading enzyme can be dried and held on a component such as a sample pad (sample supply site). When the sample is added, the IgA-specific degrading enzyme dissolves and unfolds into a solid phase, thus becoming present in the reaction system.

[0081] In this invention, the concentration of the IgA-specific degrading enzyme is only required to be such that it does not strongly affect the antigen-antibody reaction between the analyte and the specific binding partner, and can exert the desired non-specific reaction inhibition effect. Those skilled in the art can set the concentration appropriately according to the analyte and the type of sample.

[0082] The concentration of the IgA-specific degrading enzyme in the antigen-antibody reaction system varies depending on the reagent composition of the system. For example, when adding an IgA-specific degrading enzyme to the sample pretreatment solution, 1–1000 U, preferably 5–800 U, more preferably 10–500 U, further preferably 15–300 U, and most preferably 20–200 U can be added to 10 μL of sample. Furthermore, when adding an IgA-specific degrading enzyme to the sample pretreatment solution, the enzyme concentration can be 1–1000 U / μL, preferably 5–800 U / μL, more preferably 10–500 U / μL, further preferably 15–300 U / μL, and most preferably 20–200 U / μL. In addition, when an IgA-specific degrading enzyme is added to the sample pretreatment solution, the mixing ratio of the sample to the sample pretreatment solution can be 1:100 to 100:1, preferably 1:50 to 50:1, more preferably 1:20 to 20:1, and most preferably 1:10 to 10:1.

[0083] In this invention, the IgA-specific degrading enzyme can be used alone or in combination with other substances that have non-specific reaction-inhibiting effects. Examples of other substances with non-specific reaction-inhibiting effects include anti-IgA antibodies, polymeric compounds, Heteroblock (manufactured by OMEGA Biologicals), and modified antibodies that alter part or all of the variable regions in the L or H chains of a specific antibody. It should be noted that other substances with non-specific reaction-inhibiting effects are not limited to those listed herein. By using the IgA-specific degrading enzyme in combination with other substances with non-specific reaction-inhibiting effects, an enhancement of the non-specific reaction-inhibiting effect is expected. Furthermore, when using both anti-IgA antibodies and IgA-specific degrading enzymes, the anti-IgA antibody and the enzyme can be directly or indirectly combined.

[0084] When the immunoassay reagent of the present invention contains an IgA-specific degrading enzyme in advance, it is preferable that the enzyme is contained in the assay reagent in such a way that it reaches the concentration in the above-described reaction system.

[0085] [Latex Immunoturbidimetric Assay (LTIA)]

[0086] The LTIA method, which is one of the immunological assay methods of the present invention, will be described. Methods for measuring the target substance using the LTIA method can be broadly classified into two types.

[0087] The first method is as follows: latex particles immobilized with a specific binding coupler to the analyte react with the analyte to form a sandwich-type immune complex, and the analyte is measured based on the degree of aggregation of the latex particles accompanying the formation of the immune complex.

[0088] The second method is as follows: Proteins or similar substances immobilized with multiple analyte materials or their analogues (including fragments thereof) are pre-added to the immunoassay reagent. These proteins compete with the analyte material in the sample, hindering the formation of an immune complex between the analyte material contained in the reagent and latex particles immobilized with a specific binding partner for the analyte material. The analyte material (e.g., antigen, etc.) is measured based on the degree of inhibition of latex particle aggregation accompanying the inhibition of immune complex formation.

[0089] The analyte and its specific binding partner can be any of the following: protein, peptide, glycan, lipid, glycoprotein, glycolipid, nucleic acid, low-molecular-weight compound, or high-molecular-weight compound, as long as they can specifically bind to the analyte. Any substance can be selected depending on the purpose. For example, if the analyte is an antigen, any antibody, such as polyclonal antibodies or monoclonal antibodies (including monoclonal antibodies produced by hybridomas, recombinant antibodies, and functional fragments of various antibodies), can be selected as the specific binding partner. If the analyte is an antibody, antigens such as natural and recombinant antigens can be selected as the specific binding partner.

[0090] The present invention can be used in any of the above methods. Specifically, the following steps can be exemplified, but are not limited thereto.

[0091] (1) The process of contacting the sample containing the substance to be measured with the IgA-specific degrading enzyme in solution.

[0092] (2) A step after step (1) of adding latex particles carrying a specific binding coupler for the analyte to the solution.

[0093] (3) A step following step (2) to optically detect the degree of aggregation of the latex particles in the solution.

[0094] Here, (3) refers to "the process of determining the agglomeration reaction between the test object and the latex particles without going through the washing and separation process, either in the middle of (2) or after (2)".

[0095] LTIA (Low-Temperature Interference Analysis) allows for the determination of the analyte by optically observing the degree of agglomeration. Examples of optical observation methods include measuring the intensity of scattered light, absorbance, or transmitted light intensity using optical equipment (endpoint method, rate method, etc.). For the changes in absorbance, etc., measured in this way, a standard curve is obtained by measuring with a standard substance whose concentration of the analyte is known, and the concentration (quantitative value) of the analyte in the sample is calculated. It should be noted that the measurement of absorbance, etc., of transmitted or scattered light can be performed using a single wavelength or two wavelengths (based on the difference or ratio of the two wavelengths). The measurement wavelength is typically selected from 500 nm to 900 nm.

[0096] In this invention, the determination of the target substance in the sample can be performed manually or using a measuring device. The measuring device can be a general-purpose automatic analytical device or a dedicated measuring device (special-purpose machine). Furthermore, the determination is preferably performed using a method involving multiple operational steps, such as a two-step method (two-reagent method).

[0097] [Latex particles carrying specific binding couplers]

[0098] Specific binding pairs for the analyte can be immobilized and loaded onto latex particles using known methods such as physical adsorption, chemical binding, or a combination thereof. In the case of physical adsorption, this can be performed by mixing and contacting the specific binding pair for the analyte and latex particles in a solution such as a buffer solution, or by contacting the specific binding pair for the analyte dissolved in a buffer solution with a carrier, etc. Furthermore, when using chemical bonding methods, it is possible to follow the guidelines outlined in "Clinical Pathology Temporary Supplement No. 53: Immunoassay for Clinical Examination - Techniques and Applications" (published by the Japanese Society for Clinical Pathology, 1983); and "New Chemical Experiment Lecture 1: Tanpaku Quality IV" (published by the Japanese Biochemical Society). The known method described in "Protein IV", published by Tokyo Chemical Dojin in 1991, involves mixing and contacting a specific binding partner and carrier for the target substance with a divalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide, and reacting the amino, carboxyl, thiol, aldehyde, or hydroxyl groups of the specific binding partner and carrier with the divalent crosslinking reagent.

[0099] The synthetic polymer constituting the latex particles is not particularly limited, and examples include polystyrene, styrene-styrene sulfonate copolymers, methacrylic acid polymers, acrylic acid polymers, itaconic acid polymers, and styrene-hydrophilic carboxyl monomer copolymers. Examples include styrene-methacrylic acid copolymers, styrene-acrylic acid copolymers, and styrene-itaconic acid copolymers. Among these, styrene-methacrylic acid copolymers, styrene-itaconic acid copolymers, and styrene and styrene-styrene sulfonate copolymers are preferred. Styrene and styrene-(meth)acrylic acid copolymers are particularly preferred.

[0100] Regarding the specific binding coupler for the analyte carried on the latex particles, multiple couplers are preferred to form a sandwich structure. When multiple antibody recognition sites exist in the analyte, only one specific binding coupler may be used. For example, when the specific binding coupler is a monoclonal antibody, multiple monoclonal antibodies with different recognition sites can be used. Alternatively, when the specific binding coupler is a polyclonal antibody, it can be a polyclonal antibody derived from one antiserum or from multiple antiserums. Furthermore, monoclonal antibodies and polyclonal antibodies can be used in combination.

[0101] If treatment is required to suppress the natural aggregation and non-specific reactions of latex particles, it can be carried out by known methods such as coating the surface of latex particles with proteins such as bovine serum albumin (BSA), casein, gelatin, ovalbumin or its salts, surfactants or skim milk powder, to achieve carrier sealing treatment (masking treatment).

[0102] [Reagents for Immunological Assays]

[0103] The immunological assay method of the present invention can be performed using immunological assay reagents. The immunological assay reagents are characterized in that, in addition to the main component of the antigen-antibody reaction, they also contain the aforementioned IgA-specific degrading enzyme. Examples of the main component include specific binding couplers for the analyte substance; further examples include immunological assay particles, immunochromatographic test strips, microplates, and other insoluble carriers.

[0104] The immunoassay reagent of the present invention may contain buffers, proteins, peptides, amino acids, nucleic acids, lipids, phospholipids, carbohydrates, glycoproteins, glycolipids, inorganic salts, polymers, surfactants, other non-specific reaction inhibitors, preservatives, etc., within a range that does not interfere with the non-specific reaction inhibition effect of IgA-specific degrading enzymes. Components for buffering and adjusting the pH, ionic strength, osmotic pressure, etc., of the sample may include, for example, buffers such as acetic acid, citric acid, phosphoric acid, Tris, glycine, boric acid, carbonic acid, phthalic acid, succinic acid, maleic acid, imidazole, etc., as well as GOOD'S buffer, and their sodium, potassium, and calcium salts. Furthermore, components that enhance the aggregation of particles for immunoassay may also include polymers such as polyvinylpyrrolidone and phospholipid polymers.

[0105] Regarding the concentration of IgA-specific degrading enzyme in the reagent, it should be a concentration that can be adjusted to the concentration within the antigen-antibody reaction system under the mixed state of the reagent and sample during the assay, which varies depending on the type of reagent.

[0106] [Immunological Assay Kit]

[0107] The immunoassay kit of the present invention is characterized in that it contains at least an IgA-specific degrading enzyme in its composition. Therefore, as a kit of the present invention, in addition to reagents related to the antigen-antibody reaction constituting the kit, it contains an IgA-specific degrading enzyme in any one or more of the following: sample diluent, sample extract, etc. In addition to the above, examples of kit composition include an instruction manual and sample collection equipment (collection pipettes, syringes, cotton swabs, filters, etc.).

[0108] The following sections describe the reagent composition for each immunological assay method.

[0109] [Latex Immunoturbidimetric Method]

[0110] Examples of reagents (LTIA reagents) used in immunological assays where the method is latex immunoturbidimetric assay are given, but not limited to these examples.

[0111] (1) First reagent containing IgA-specific degrading enzyme

[0112] (2) A second reagent comprising latex particles carrying specific binding couplers for the analyte.

[0113] The first reagent typically includes a buffer solution. The concentration of the IgA-specific enzyme in the buffer solution is sufficient to adjust to the preferred enzyme concentration when the reagent and sample are mixed during the assay; this varies depending on the type of reagent. The enzyme may also be included in a second reagent in addition to the first.

[0114] In an example of the LTIA reagent of the present invention, the concentration of the IgA-specific degrading enzyme contained in the conventional first reagent is 1 to 1000 μg / mL, preferably 5 to 500 μg / mL, more preferably 10 to 100 μg / mL, and even more preferably 20 to 50 μg / mL, but is not limited to this concentration.

[0115] When adding a sample pretreatment solution to the sample before supplying it to the LTIA method, the IgA-specific degrading enzyme may also be included in the sample pretreatment solution, wherein the concentration of the enzyme in the pretreatment solution is 1 to 1000 μg / mL, preferably 5 to 500 μg / mL, more preferably 10 to 300 μg / mL, and even more preferably 20 to 200 μg / mL, but is not limited to this concentration.

[0116] (temperature)

[0117] Regarding the reaction temperature of the IgA-specific degrading enzyme for the sample, it is acceptable as long as the IgA-specific degrading enzyme will not be inactivated and can specifically degrade IgA at a temperature ranging from 0 to 60°C, preferably from 10 to 50°C, and more preferably from 20 to 40°C, but is not limited to this temperature.

[0118] (Reaction time)

[0119] Regarding the reaction time of the IgA-specific degrading enzyme for the sample, it is acceptable as long as the reaction time is sufficient to specifically and fully degrade IgA using the IgA-specific degrading enzyme, ranging from 30 seconds to 24 hours, preferably from 1 minute to 120 minutes, but not limited to this reaction time.

[0120] (pH)

[0121] The pH of the solution containing the IgA-specific degrading enzyme is only required to allow the IgA-specific degrading enzyme to function fully, and is in the range of pH 1 to 12, preferably 3 to 10, more preferably 5 to 9, and most preferably 6 to 8, but is not limited to this pH. It should be noted that compared to known protein-degrading enzymes such as papain, pepsin, and trypsin, the IgA-specific degrading enzyme has a wider pH range for increasing enzyme activity.

[0122] (Particles for immunological assay)

[0123] In addition to the latex particles mentioned above, any known particles can be used for immunoassays in this invention as long as they can carry a specific binding pair for the analyte. For example, inorganic particles such as metal colloids, silica, carbon, and magnetic particles can also be used as the immunoassay particles of this invention.

[0124] The particle size for immunological assays can be appropriately selected from the range of 0.05 to 1 μm, taking into account the optical assay method used (e.g., turbidimetry for measuring transmitted light, turbidimetry for measuring scattered light, etc.), to obtain the desired assay sensitivity, assay range, etc. It should be noted that in optical assays of automated analytical devices, an average particle size of 0.1 to 0.4 μm is commonly used, but not limited to this range.

[0125] [ELISA method]

[0126] ELISA is a method that utilizes various combinations of antigen-antibody reactions, ultimately embedding enzyme-labeled antigens or antibodies into the reaction system to detect enzyme activity. Enzyme activity detection uses substrates whose absorbance spectra change due to the reaction. Depending on the combination of antigen-antibody reactions, methods such as direct methods, indirect methods, sandwich methods, and competitive methods can be categorized.

[0127] Examples of immunoassay reagents are provided for the case where the immunoassay method of the present invention is a sandwich ELISA method.

[0128] (a) An insoluble carrier immobilized with antibodies that react with the analyte.

[0129] (b) Antibodies labeled with a labeling substance that react with the analyte.

[0130] As the insoluble carrier in (a), a plate is preferred, and the labeling substance can be appropriately selected. An antibody immobilized on the insoluble carrier captures the analyte in a solution containing the sample, forming a complex on the insoluble carrier. The antibody labeled with the labeling substance binds to the captured analyte, forming a sandwich with the aforementioned complex. By determining the amount of the labeling substance according to the labeling method, the analyte in the sample can be determined. Regarding specific methods such as the method of antibody immobilization on the insoluble carrier and the method of antibody binding with the labeling substance, methods known to those skilled in the art can be used without particular limitation.

[0131] In the ELISA method, the IgA-specific degrading enzyme of the present invention may be present in the immune reaction system, for example, by adding it to the sample pretreatment solution or to the solution in which the antigen-antibody reaction is performed.

[0132] [Immunochromatography]

[0133] The composition (test strip composition) of the reagents for immunoassay in the case where the immunoassay method of the present invention is immunochromatography will be described.

[0134] Immunochromatographic test strips;

[0135] When using antibodies as specific binding partners, a test sheet is constructed on a sheet-like insoluble carrier such as a porous membrane, with "1. a sample supply site", "2. a site for holding the labeled antibody (labeled antibody holding site)" and "3. a site for immobilizing the antibody used to capture the complex formed by the labeled antibody and the analyte (capture antibody site)" sequentially along the unfolding direction of the solution containing the sample.

[0136] In immunochromatography, at least the test strip described above is included. If a predetermined amount of a sample containing the analyte is added to the sample supply site, the sample invades the labeled antibody holding site via capillary action. The analyte binds to the labeled antibody to form a complex. If this complex expands the membrane and invades the capture antibody site, it is captured by the antibody immobilized on the membrane (capture antibody), forming a capture antibody-analyte-labeled antibody complex. The analyte can then be detected by detecting the label using any method (e.g., agglutination image in the case of a visually perceptible label such as gold colloid, or a colorimetric reaction caused by substrate addition in the case of an enzyme).

[0137] In immunochromatography, the IgA-specific degrading enzyme of the present invention can be present in the reaction system, for example, by adding it to a sample pretreatment solution or by including it in a sample supply site or a labeled antibody holding site and drying it.

[0138] Chemiluminescence method

[0139] This method involves reacting magnetic particles bound to antigens or antibodies with the analyte to form a complex, then removing unreacted substances using magnetic force. Another method involves adding reagents containing labeled antibodies, removing unreacted substances using magnetic force, adding a luminescent reagent, and measuring the amount of light emitted. When enzymes are used as labels, it is called chemiluminescent enzyme immunoassay (CLEIA). When metal complexes such as ruthenium-pyridine complexes are used as labels and the light intensity is measured through an electrochemical reaction, it is called electrochemiluminescence immunoassay (ECLIA). Finally, when chemiluminescent substances are used as labels, it is called chemiluminescent immunoassay (CLIA).

[0140] Examples of immunoassay reagents are provided for the CLEIA method of the present invention.

[0141] (a) Immobilized magnetic particles containing antibodies (or antigens) that react with the analyte.

[0142] (b) Antibodies (or antigens) labeled with enzymes and reacting with the substance to be measured.

[0143] (c) Luminescent reagent

[0144] Antibodies immobilized on magnetic particles capture the analyte in a solution containing the sample, forming a complex. An antibody labeled with an enzyme-labeled substance binds to the captured analyte, forming a sandwich with the complex. By reacting the enzyme-labeled substance with a luminescent reagent to measure the amount of luminescence, the analyte in the sample can be determined.

[0145] In the CLEIA method, the IgA-specific degrading enzyme of the present invention may be present in the immune reaction system, for example, by adding it to the sample pretreatment solution or by adding it to the solution in which the antigen-antibody reaction is carried out.

[0146] [Specific binding to mating bodies]

[0147] In this invention, the specific binding partner for the analyte can be any substance capable of specifically binding to the analyte, including proteins, peptides, amino acids, lipids, carbohydrates, glycoproteins, glycolipids, nucleic acids, haptens, low-molecular-weight compounds, and high-molecular-weight compounds. Furthermore, there are no particular limitations on molecular weight or source (natural or synthetic), and examples of antibodies or antigens suitable for use in immunological assays utilizing antigen-antibody reactions can be provided.

[0148] The antibody can be a polyclonal antibody or a monoclonal antibody. Monoclonal antibodies are more preferred.

[0149] <Sample>

[0150] In this invention, examples of samples containing the substance to be measured include human or animal blood, serum, plasma, culture supernatant, urine, bone marrow fluid, saliva, sweat, ascites, nasal discharge, feces, or cell or tissue extracts. Blood is most preferably used as the sample containing the substance to be measured. It should be noted that the standard value for IgA in blood is typically 110–410 mg / dL. The sample is sometimes referred to as a "sample".

[0151] [Substance to be measured]

[0152] The immunological assay reagent of the present invention can use various substances contained in the sample as the analyte. Examples of analytes include proteins, peptides, amino acids, lipids, carbohydrates, glycoproteins, glycolipids, nucleic acids, haptens, etc., and there are no particular limitations on any theoretically measurable substance. Examples include C-reactive protein (CRP), lipoprotein(a) (Lp(a)), matrix metalloproteinase 3 (MMP3), antiphospholipid antibodies, type IV collagen, prostate-specific antigen (PSA) (molecular weight: 34000), brain natriuretic peptide (BNP) (molecular weight: 3500), and N-terminal probrain natriuretic peptide (NT-pro) BNP (molecular weight: 8500), insulin (molecular weight: 5800), albumin, cystatin C, rheumatoid factor (RF), KL-6, procalcitonin (PCT) (molecular weight: 13000), fibrin and fibrinogen breakdown products (FDP), D-dimer, soluble fibrin (SF), thrombin-antithrombin III complex (TAT), transferrin, haptoglobin, α1-antitrypsin, α1-acid glycoprotein, α2-macroglobulin, hemoglobin-binding protein, antithrombin-III, α-alpha-fetoprotein, carcinoembryonic antigen (CEA), ferritin, hepatitis B surface antigen (HBsAg) g), anti-hepatitis B surface antigen (HBs), anti-hepatitis B e antigen (HBe-Ag), anti-hepatitis B e antigen antibody (Anti-HBe), anti-hepatitis B core antibody (Anti-HBc), severe acute respiratory syndrome virus (SARS), PAI-1 (molecular weight: 42700), phenytoin, phenobarbital, carbamazepine, valproic acid, theophylline, thymic activating regulatory chemokine (TARC) (molecular weight: 8100), soluble interleukin-2 receptor (sIL-2R) (molecular weight: 45000), and pulmonary surfactant protein D (SP-D), etc. The molecular weight of the target substance can be below 50000, preferably below 50000.

[0153] Examples of substances that can be measured in this invention include antibodies against Treponema pallidum, cyclic citrullinated peptide (CCP), Helicobacter pylori, and IgG and IgM antibodies against viruses such as hepatitis, measles, and leukemia. In the technology described in Patent Document 2, these antibodies may be decomposed by a reducing agent. However, the IgA-specific decomposing enzyme in this invention does not decompose these antibodies. Therefore, the IgA-specific decomposing enzyme is particularly useful for the technology described in Patent Document 2 when the analyte is an antibody.

[0154] [Non-specific reaction inhibitors]

[0155] In this invention, inhibiting nonspecific reactions refers to the action of factors (also called nonspecific factors, nonspecific causal substances, or nonspecific reactive substances) on the aforementioned nonspecific reactions occurring in biological samples, inhibiting the influence of reactions other than antigen-antibody reactions on the assay. Therefore, in this invention, whether a candidate substance, as a nonspecific reaction inhibitor, has an inhibitory effect on nonspecific reactions can be determined, for example, by using the measured value (hereinafter referred to as the control method measured value) obtained through a assay method with B / F separation (a method with a washing step that is less likely to produce the influence of nonspecific reactive substances (CLEIA method in the examples)) as a benchmark, and by comparing the value obtained with the addition of the candidate substance to the value obtained without the addition of the candidate substance to the value obtained through the control method measured value. That is, in the target assay method, if the measured value obtained with the addition of the candidate substance is closer to the control method measured value than the measured value obtained without the addition of the candidate substance, it can be determined that the candidate substance has an inhibitory effect on nonspecific reactions in the assay method, and the candidate substance can be considered a nonspecific reaction inhibitor.

[0156] The nonspecific reaction inhibitor of the present invention targets both factors that produce positive measurement errors (i.e., factors that determine the content of the target substance to be higher than the original content) and negative measurement errors (i.e., factors that determine the content to be lower than the original content) due to certain components contained in the biological sample. It is particularly effective against nonspecific factors that cannot be suppressed by commercially available nonspecific reaction inhibitors such as HBR-1 and Heteroblock. Furthermore, it is effective against nonspecific factors that produce positive measurement errors (i.e., abnormally high measurement values ​​deviating from the sample) and negative measurement errors (i.e., abnormally low measurement values). In the present invention, various causative substances can be cited as the causative substances causing nonspecific reactions; preferably, causative substances that decompose at least a portion of IgA, all of IgA, or have a structure similar to a portion or all of IgA are preferred. Through this decomposition reaction, the nonspecific reaction inhibitor of the present invention can suppress nonspecific reactions originating from this causative substance.

[0157] The nonspecific reaction inhibitor of the present invention only needs to contain a substance as determined above that is capable of inhibiting the reaction caused by nonspecific factors originating from the sample, and at least contains an IgA-specific degrading enzyme as an active ingredient. The nonspecific reaction inhibitor of the present invention can be configured to contain an IgA-specific degrading enzyme in the above-described immunoassay reagent.

[0158] In the non-specific reaction inhibitor of the present invention, within the range that does not interfere with the non-specific reaction inhibitory effect of the IgA-specific degrading enzyme, it may contain a buffer, protein, peptide, amino acid, nucleic acid, lipid, phospholipid, saccharide, glycoprotein, glycolipid, inorganic salt, high molecular compound, surfactant, other non-specific reaction inhibitors, preservative, etc. As other non-specific reaction inhibitors, as long as they have a non-specific reaction inhibitory effect, anti-IgA antibody, high molecular compound, altered antibody in which a part or all of the variable region in the L chain or H chain of a specific antibody is altered, etc. can be cited, but not limited thereto. By using the IgA-specific degrading enzyme in combination with other enzymes having a non-specific reaction inhibitory effect, an enhanced non-specific reaction inhibitory effect can be expected. Further, in the case of using an anti-IgA antibody and an IgA-specific degrading enzyme in combination, it may also be in a state where the anti-IgA antibody and the enzyme are directly or indirectly bound.

[0159] [Method for inhibiting non-specific reaction]

[0160] The non-specific reaction inhibition method of the present invention refers to a method for inhibiting non-specific reactions caused by a sample by performing at least one or more antigen-antibody reactions in the presence of an IgA-specific degrading enzyme. In addition, the non-specific reaction inhibition method can also be referred to as a method for reducing measurement errors.

[0161] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to the following examples.

[0162] Examples

[0163] [Inhibition of non-specific reaction in LTIA method: Determination of sIL-2R]

[0164] The concentration of soluble interleukin 2 receptor (sIL-2R) contained in a sample was measured by the LTIA method as follows.

[0165] As samples, human serum samples 1 to 3 from multiple persons (3 persons) were used. Sample 1 (control sample) is a sample whose measured value based on the LTIA method shows a value close to the measured value based on the chemiluminescent enzyme immunoassay (CLEIA method) (Reference Example 1). Samples 2 and 3 (deviating samples) exhibit non-specific reactions, and the measured values based on the LTIA method deviate significantly from the measured value of Reference Example 1 based on the CLEIA method. It should be noted that the IgA concentration in each sample is 692 mg / dl in Sample 1, 387 mg / dl in Sample 2, and 247 mg / dl in Sample 3. The IgA concentrations of these samples were measured using the reagent of "N-Assay TIAIgA-SH Nittobo" with a Hitachi 7180 automatic analyzer.

[0166] [Reference Example 1] Determination using the CLEIA method

[0167] 1. Measurement Method

[0168] 1-1. Test reagents

[0169] Deteminer CL (registered trademark) IL-2R NX (Minaris Medical Co., Ltd.)

[0170] 1-2. Sample

[0171] Samples 1-3

[0172] 1-3. Measurement Procedure

[0173] The determination was performed using CL-JACK NX (registered trademark) (Minaris Medical Co., Ltd.) according to the accessories of the assay reagent.

[0174] 2. Measurement Results

[0175] The measurement results are shown in Table 1.

[0176] The CLEIA method shown in Reference Example 1 performs a B / F separation operation and includes a cleaning step. Therefore, the CLEIA method is a determination method that is not easily affected by non-specific reactions originating from the sample.

[0177] [Comparative Example 1] Determination using the LTIA method: PBS buffer (without additives) was added to the sample.

[0178] 1. Measurement Method

[0179] 1-1. Test reagents

[0180] The first and second reagents were prepared according to the method described in Japanese Patent Application Publication No. 2017-181377.

[0181] 1-2. Sample pretreatment solution

[0182] Use PBS buffer (pH 7.4).

[0183] 1-3. Sample

[0184] For samples (serum) 1-3 described in Reference Example 1, PBS buffer as a sample pretreatment solution was added at a volume ratio of 5:1, and the samples were measured after reacting at 37°C for 1 hour.

[0185] 1-4. Measurement Procedure

[0186] The sample, first reagent, and second reagent were mixed, and the concentration of sIL-2R in the sample was determined using a Hitachi 7180 automated analytical apparatus. Specifically, 120 μL of the first reagent was added to 5.6 μL of the sample, and the mixture was incubated at 37°C for 5 minutes. Then, 40 μL of the second reagent was added and stirred. Subsequently, the absorbance change accompanying agglomeration was measured at a dominant wavelength of 570 nm and a secondary wavelength of 800 nm over 5 minutes. This absorbance change was applied to a standard curve obtained by measuring a standard substance of known concentration, and the measured value was calculated. Furthermore, the original solution conversion value, taking into account the dilution factor of the sample, was calculated.

[0187] 2. Measurement Results

[0188] The results, converted to the values ​​per 1 mL of serum before adding the sample pretreatment solution, are shown in Table 1. The units of the test results are U / mL.

[0189] [Example 1] Determination using LTIA method: IgA-specific degrading enzyme was added to the sample.

[0190] PBS buffer containing 40 U / μL IgASAP (manufactured by Genovis) was used as the sample pretreatment solution. Otherwise, the assays were performed using the same method as in Comparative Example 1. The results are shown in Table 1.

[0191] [Table 1]

[0192]

[0193] Based on the results of Reference Example 1, Comparative Example 1, and Example 1, the non-specific reaction inhibition effect of IgA-specific degrading enzymes was investigated.

[0194] (1) The results of the determination of the control sample (sample 1) were verified.

[0195] In the control samples, the measured values ​​of Reference Example 1, Comparative Example 1, and Example 1 were approximately equivalent. These results indicate that the addition of IgASAP had no effect on the measured values ​​of samples that did not exhibit a nonspecific reaction.

[0196] (2) The measurement results that deviated from the samples (samples 2 and 3) were verified.

[0197] The measured value of Sample 2 was 738 U / mL in Reference Example 1. Comparative Example 1 was 1419 U / mL, deviating from the measured value of Reference Example 1. On the other hand, the measured value of Example 1 was 810 U / mL, showing a tendency to be close to that of Reference Example 1.

[0198] Regarding Sample 3, the same tendency was also obtained. The measured value of Sample 3 was 630 U / mL in Reference Example 1. In Comparative Example 1, it was 1766 U / mL, deviating from the measured value of Reference Example 1. On the other hand, the measurement result of Example 1 was 756 U / mL, showing a tendency to be close to that of Reference Example 1.

[0199] As described above, in the immunological assay method, by using an IgA-specific degrading enzyme, it is possible to inhibit the non-specific reaction derived from the sample. The deviated sample implemented in this test is a sample in which sufficient non-specific reaction inhibition effect cannot be obtained by only using existing non-specific reaction inhibitors such as anti-IgA antibody. By using the non-specific reaction inhibition method of the present invention using an IgA-specific degrading enzyme, it is possible to inhibit the non-specific reaction for the first time.

[0200] [Experimental Example 2]

[0201] [Inhibition of Non-Specific Reaction in LTIA Method: Measurement of sIL-2R]

[0202] Similar to Example 1, the concentration of soluble interleukin 2 receptor (sIL-2R) contained in the test sample (sample) was measured by the LTIA method. The test sample used was human serum sample 2.

[0203] [Comparative Example 2] Measurement by LTIA Method: Adding PBS Buffer (No Additive) to the Test Sample

[0204] 1. Measurement Method

[0205] 1-1. Measurement Reagent

[0206] According to the method described in JP-A-2017-181377, the first reagent and the second reagent were prepared.

[0207] 1-2. Sample Pretreatment Solution

[0208] PBS buffer (pH 7.4) was used.

[0209] 1-3. Test Sample

[0210] To the sample (serum) 2 described in Reference Example 1, PBS buffer as the sample pretreatment solution was added at a volume ratio of 4.5:1.5, and the test sample after reacting at 37°C for 1 hour was measured.

[0211] 1-4. Measurement Procedure

[0212] The sample, first reagent, and second reagent were mixed, and the concentration of sIL-2R in the sample was determined using a Hitachi 7180 automated analytical apparatus. Specifically, 120 μL of the first reagent was added to 5.6 μL of the sample, and the mixture was incubated at 37°C for 5 minutes. Then, 40 μL of the second reagent was added and stirred. Subsequently, the absorbance change accompanying agglomeration was measured at a dominant wavelength of 570 nm and a secondary wavelength of 800 nm over 5 minutes. This absorbance change was applied to a standard curve obtained by measuring a standard substance of known concentration, and the measured value was calculated. Furthermore, the original solution conversion value, taking into account the dilution factor of the sample, was calculated.

[0213] 2. Measurement Results

[0214] The results, converted to the values ​​per 1 mL of serum before adding the sample pretreatment solution, are shown in Table 2. The units of the test results are U / mL.

[0215] [Reference Example 2] Determination using LTIA method: Add a commercially available nonspecific reaction inhibitor (Heteroblock) to the sample.

[0216] PBS buffer containing 3.3 mg / mL Heteroblock (manufactured by OMEGA Biologicals) was used as the sample pretreatment solution. Otherwise, the assays were performed using the same method as in Comparative Example 2. The results are shown in Table 2.

[0217] [Example 2] Determination using LTIA method: IgA-specific degrading enzyme was added to the sample.

[0218] PBS buffer containing 26.7 U / μL of IgASAP (manufactured by Genovis) was used as the sample pretreatment solution. Otherwise, the assays were performed using the same method as in Comparative Example 2. The results are shown in Table 2.

[0219] [Comparative Example 3] Determination using LTIA method: Pepsin was added to the sample.

[0220] The sample pretreatment solution was PBS buffer containing 3.3 mg / mL Pepsin (manufactured by Roche, dissolved in 0.2 M citrate buffer, pH 3.0). Otherwise, the assays were performed using the same method as in Comparative Example 2. The results are shown in Table 2.

[0221] [Example 3] Determination using LTIA method: IgA-specific degrading enzyme and commercially available non-specific reaction inhibitor (Heteroblock) were added to the sample.

[0222] PBS buffer containing 26.7 U / μL IgASAP (manufactured by Genovis) and 3.3 mg / mL Heteroblock (manufactured by OMEGA Biologicals) was used as the sample pretreatment solution. Otherwise, the assays were performed using the same method as in Comparative Example 2. The results are shown in Table 2.

[0223] [Comparative Example 4] Determination using LTIA method: Pepsin and a commercially available nonspecific reaction inhibitor (Heteroblock) were added to the sample.

[0224] Sample pretreatment was performed using PBS buffer containing 3.3 mg / mL Pepsin (manufactured by Roche, dissolved in 0.2 M citrate buffer, pH 3.0) and 3.3 mg / mL Heteroblock (manufactured by OMEGA Biologicals). Otherwise, the assays were performed using the same method as in Comparative Example 2. The results are shown in Table 2.

[0225] [Table 2]

[0226]

[0227] Based on the results of Reference Example 1, Reference Example 2, Comparative Examples 2, 3, 4, and Examples 2 and 3, the inhibitory effects of IgA-specific degrading enzymes, non-specific reactions of Pepsin, and their combined effects with commercially available non-specific reaction inhibitors (Heteroblock) were investigated.

[0228] (1) Verify the effects of IgA-specific degrading enzyme and Pepsin.

[0229] The measured value of Sample 2 was 738 U / mL in Reference Example 1. Comparative Example 2 (without additive) was 2092 U / mL, deviating from Reference Example 1. Example 2 (with added IgA-specific degrading enzyme) had a measured value of 977 U / mL, showing a tendency to approach Reference Example 1. On the other hand, Comparative Example 3 (with added Pepsin) had a measured value of 2135 U / mL, deviating from Reference Example 1, and was at the same level as Comparative Example 2 (without additive), indicating no confirmation of non-specific reaction inhibition.

[0230] (2) The combined effect of Pepsin and a commercially available nonspecific reaction inhibitor (Heteroblock) was verified.

[0231] Regarding the measured value of sample 2, Reference Example 2 (with added Heteroblock) was 1180 U / mL, while Comparative Example 4 (with both Pepsin and Heteroblock) was 1271 U / mL, showing a tendency to deviate from Reference Example 1. This suggests that Pepsin is difficult to use in combination with other nonspecific inhibitors.

[0232] (3) The combined effect of IgA-specific degrading enzyme and commercially available non-specific reaction inhibitor (Heteroblock) was verified.

[0233] Regarding the measured values ​​of Sample 2, Reference Example 2 (with Heteroblock added) was 1180 U / mL, and Example 2 (with IgA-specific degrading enzyme added) was 977 U / mL. Compared with Comparative Example 2 (without additives), the measured values ​​were lower, showing considerable improvement, but there is still room for improvement compared to the measured values ​​of Reference Example 1. On the other hand, the measured value of Example 3 (using both IgA-specific degrading enzyme and Heteroblock) was 908 U / mL, showing a tendency to be closer to Reference Example 1. This suggests that IgA-specific degrading enzymes can be used in combination with other non-specific reaction inhibitors, and the effect is enhanced by further combination.

[0234] As described above, in immunological assays, non-specific reactions originating from the sample can be inhibited by using IgA-specific degrading enzymes. Furthermore, non-specific reaction inhibition was also observed in deviation samples where the addition of Pepsin did not confirm a sufficient effect. Therefore, IgA-specific degrading enzymes can be used in conjunction with commercially available non-specific reaction inhibitors, and their usefulness is significantly higher than that of Pepsin.

[0235] The off-samples used in this experiment were samples for which existing non-specific reaction inhibitors, such as anti-IgA antibodies, could not achieve sufficient inhibition. By using the non-specific reaction inhibition method of the present invention, which utilizes IgA-specific degrading enzymes, non-specific reactions were inhibited for the first time.

Claims

1. An immunological assay method for determining the analyte in a sample, characterized in that, In this immunological assay method, In the presence of an enzyme that specifically breaks down immunoglobulin A, at least one antigen-antibody reaction occurs.

2. The immunological assay method according to claim 1, wherein, The enzyme is a protease.

3. The immunological assay method according to claim 2, wherein, The protease is an endopeptidase or an exopeptidase.

4. The immunological assay method according to claim 1 or 2, wherein, The enzyme is an enzyme that cleaves the amino acid sequence containing the IgA constant region from CHα1 to CHα3.

5. The immunological assay method according to claim 1 or 2, wherein, The enzyme is an enzyme that acts on IgA in the sample and breaks down the IgA into Fab fragments and Fc fragments.

6. The immunological assay method according to claim 1 or 2, wherein, The IgA is IgA1.

7. The immunological assay method according to claim 1 or 2, wherein, The immunological assay method is latex immunoturbidimetric assay.

8. The immunological assay method according to claim 7, wherein, In the immunological assay method, the substance to be measured is an antigen or an antibody.

9. A reagent for an immunological assay, characterized in that, An immunological assay method for determining the analyte in a sample, wherein the reagents for the immunological assay contain an enzyme that specifically breaks down IgA.

10. The reagent for immunological assay according to claim 9, wherein, The enzyme is a protease.

11. The reagent for immunological assay according to claim 10, wherein, The protease is an endopeptidase or an exopeptidase.

12. The reagent for immunological assay according to claim 9 or 10, wherein, The enzyme is an enzyme that cleaves the amino acid sequence containing the IgA constant region from CHα1 to CHα3.

13. The reagent for immunological assay according to claim 9 or 10, wherein, The enzyme is an enzyme that acts on IgA in the sample and breaks down the IgA into Fab fragments and Fc fragments.

14. The reagent for immunological assay according to claim 9 or 10, wherein, The IgA is IgA1.

15. The reagent for immunological assay according to claim 9 or 10, wherein, The reagents used for the immunological assay are those used in latex immunoturbidimetric assays.

16. The reagent for immunological assay according to claim 15, wherein, In the immunological assay method, the substance to be measured is an antigen or an antibody.

17. A sample pretreatment solution for immunological assays, characterized in that, It contains enzymes that specifically break down IgA.

18. The sample pretreatment solution for immunological assay according to claim 17, wherein, The enzyme is a protease.

19. The sample pretreatment solution for immunological assay according to claim 18, wherein, The protease is an endopeptidase or an exopeptidase.

20. The sample pretreatment solution for immunological assays according to claim 17 or 18, wherein, The enzyme is an enzyme that cleaves the amino acid sequence containing the IgA constant region from CHα1 to CHα3.

21. The sample pretreatment solution for immunological assays according to claim 17 or 18, wherein, The enzyme is an enzyme that acts on IgA in the sample and breaks down the IgA into Fab fragments and Fc fragments.

22. The sample pretreatment solution for immunological assays according to claim 17 or 18, wherein, The IgA is IgA1.

23. The sample pretreatment solution for immunological assays according to claim 17 or 18, wherein, The sample pretreatment solution for the immunological assay is a reagent solution used in latex immunoturbidimetry.

24. A kit for immunological assay, characterized in that, An immunological assay method for determining the analyte in a sample, wherein the kit contains an enzyme that specifically breaks down IgA.

25. The immunoassay kit according to claim 24, wherein, The enzyme is a protease.

26. The immunoassay kit according to claim 25, wherein, The protease is an endopeptidase or an exopeptidase.

27. The immunoassay kit according to claim 24 or 25, wherein, The enzyme is an enzyme that cleaves the amino acid sequence containing the IgA constant region from CHα1 to CHα3.

28. The immunoassay kit according to claim 24 or 25, wherein, The enzyme is an enzyme that acts on IgA in the sample and breaks down the IgA into Fab fragments and Fc fragments.

29. The immunoassay kit according to claim 24 or 25, wherein, The IgA is IgA1.

30. A non-specific reaction inhibitor, characterized in that, It contains enzymes that specifically break down IgA.

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