Immunoassay method, immunoassay reagent, immunoassay specimen pretreatment solution, immunoassay reagent kit, and nonspecific reaction inhibitor

Abstract

An immunological assay method for measuring a substance to be measured in a sample, in which an antigen-antibody reaction is carried out at least once in the presence of an enzyme that specifically degrades immunoglobulin A, abbreviated as IgA.

Description

[Technical Field]

[0001] The present invention relates to an immunoassay method, an immunoassay reagent, a sample pretreatment solution for immunoassay, an immunoassay reagent kit, and a non-specific reaction inhibitor. This application claims priority to U.S. provisional application No. 63 / 559,187, filed February 29, 2024, the contents of which are incorporated herein by reference. [Background technology]

[0002] One measurement method in the field of diagnostic drugs is the immunological measurement method, which measures the target substance present in a biological sample by utilizing an antigen-antibody reaction. Because the immunological measurement method utilizes an antigen-antibody reaction, it is a measurement method with extremely high specificity. Various substances exist in biological samples, and measurement errors occur when substances other than the substance being measured cause nonspecific binding reactions or interfere with specific antigen-antibody reactions. Such phenomena are called nonspecific reactions, and the substances that cause nonspecific reactions are called nonspecific factors. The existence of heterophilic antibodies and rheumatoid factors (RF) has been elucidated as nonspecific factors. Heterophilic antibodies are a general term for human antibodies that show reactivity to animal-derived antibodies, which are the main reaction in immunological assays, and human anti-mouse immunoglobulin antibodies (HAMA) are known to be a representative example. Rheumatoid factors are glycoproteins that are frequently detected in patients with collagen diseases such as rheumatoid arthritis, chronic infections, and liver diseases, and share the characteristic of showing reactivity to animal-derived antibodies with HAMA. Generally, IgM-RF of the IgM class is the most frequently detected RF, but the existence of IgA-RF has also been confirmed (Non-Patent Documents 1 and 3). Furthermore, immunoglobulin A (hereinafter referred to as IgA) is known to cause non-specific reactions in immunological measurements (Non-Patent Document 2).

[0003] Techniques for suppressing non-specific reactions in immunological assays are known, for example, from Patent Documents 1, 2 and 3. Patent Document 1 discloses a method for suppressing nonspecific reactions caused by RF by treating a sample in advance with a sufficient amount of animal-derived antibodies capable of binding 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). Patent Document 2 discloses a method for suppressing nonspecific reactions caused by interfering substances, such as rheumatoid factors, which have a structure in which polypeptide chains are bonded together via disulfide bonds, by using a reducing agent to cleave the disulfide bonds and thereby decompose the interfering substance. Patent Document 3 discloses a method for reducing or eliminating interference from non-target proteins by mixing a liquid sample containing the target protein and one or more other non-target proteins with a protein digesting agent, and carrying out protein digestion under conditions that provide antigenic determinants of the target protein in the mixture. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 07-012818 [Patent Document 2] Japanese Patent Application Publication No. 13-255325 [Patent Document 3] Special Publication No. 02-021548 [Non-patent literature]

[0005] [Non-Patent Document 1] Clinical Chemistry; Vol. 23, Suppl. 175a-1 to 175a-10 (1994) [Non-patent document 2] Tokushima Red Cross Hospital Medical Journal; Vol. 18, No. 1, 56-60 (2013) [Non-patent document 3] Immunoassay. Tetsuro Kubota, Kiyotaka Fujita, Eiji Hosoi, Michiko Kajiwara. Ishiyaku Publishing Co., Ltd. P.199(2017) Summary of the Invention [Problem to be solved by the invention]

[0006] The anti-human IgM polyclonal antibody, anti-human IgG polyclonal antibody, and anti-human IgA polyclonal antibody shown in Patent Document 1 are currently used as various reagents to suppress non-specific reactions caused by natural antibodies and RF, but it has become clear that this method is not able to sufficiently suppress non-specific reactions.

[0007] The reducing agent used in Patent Document 2 to suppress nonspecific reactions caused by interfering substances with disulfide-bonded polypeptide chains may cleave disulfide bonds in the antigen or antibody to be measured or in the antigen or antibody contained in the reagent components, depending on the type and concentration, making it less versatile. Furthermore, methods using reducing agents may decompose molecules with disulfide bonds, such as IgG and IgA, without distinguishing between them. For this reason, this method could not be used, particularly when human IgG was used as the measured substance.

[0008] Examples of protein digestive agents disclosed in Patent Document 3 include pepsin, papain, and trypsin. Pepsin exhibits its enzymatic activity at an optimum pH of 1 to 3, which is in the acidic range, and therefore there is a possibility that the analyte and / or substances that specifically bind to the analyte may be denatured or decomposed. Papain and trypsin exhibit enzymatic activity against many substances, and therefore there is a possibility that the analyte and / or substances that specifically bind to the analyte may be denatured or decomposed.

[0009] An object of the present invention is to provide an immunological measurement method capable of suppressing non-specific reactions caused by IgA contained in a measurement sample. [Means for solving the problem]

[0010] In order to solve the above problems, the present inventors investigated the inhibitory effects of various substances on non-specific reactions and found that non-specific reactions can be inhibited by carrying out an antigen-antibody reaction in the presence of an enzyme that specifically degrades IgA, leading to the completion of the present invention. Specifically, the present invention has the following features.

[0011] [1] An immunological assay method for measuring a substance to be measured in a sample, in which an antigen-antibody reaction is carried out at least once in the presence of an enzyme that specifically degrades immunoglobulin A (IgA). [2] The immunological assay method according to [1], wherein the enzyme is a protease. [3] The immunological assay method according to [2], wherein the protease is a proteinase or a peptidase. [4] The immunological measurement method according to any one of [1] to [3], wherein the enzyme is an enzyme that cleaves the amino acid sequence of the IgA constant region including CHα1 to CHα3. [5] The immunological measurement method according to any one of [1] to [4], wherein the enzyme acts on IgA in the sample and decomposes the IgA into Fab fragments and Fc fragments. [6] The immunological measurement method according to any one of [1] to [5], wherein the IgA is IgA1. [7] The immunological measurement method according to any one of [1] to [6], wherein the immunological measurement method is a latex immunoturbidimetric method. [8] The immunological measurement method according to any one of [1] to [7], wherein the substance to be measured is an antigen or an antibody. [9] A reagent for immunoassay used in an immunoassay method for measuring a substance to be measured in a sample, the reagent comprising an enzyme that specifically decomposes IgA.

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

[11] The reagent for immunoassay according to

[10] , wherein the protease is a proteinase or a peptidase.

[12] The reagent for immunological assay according to any one of [9] to

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

[13] The reagent for immunological assay according to any one of [9] to

[12] , wherein the enzyme acts on IgA in the sample and decomposes the IgA into an Fab fragment and an Fc fragment.

[14] The reagent for immunological assay according to any one of [9] to

[13] , wherein the IgA is IgA1.

[15] The reagent for immunological assay according to any one of [9] to

[14] , which is a reagent for use in latex immunoturbidimetry.

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

[15] , wherein in the immunoassay method, the substance to be measured is an antigen or an antibody.

[17] A sample pretreatment solution for immunoassays, containing an enzyme that specifically degrades IgA.

[18] The sample pretreatment solution for immunological measurements according to

[17] , wherein the enzyme is a protease.

[19] The sample pretreatment solution for immunological assays according to

[18] , wherein the protease is a proteinase or a peptidase.

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

[17] to

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

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

[17] to

[20] , wherein the enzyme acts on IgA in the sample and decomposes the IgA into Fab fragments and Fc fragments.

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

[17] to

[21] , wherein the IgA is IgA1.

[23] The specimen pretreatment solution for immunological measurements according to any one of

[17] to

[22] , which is a chemical solution for use in latex immunoturbidimetry.

[24] A reagent kit for immunological measurement used in an immunological measurement method for measuring a substance to be measured in a sample, the reagent kit comprising an enzyme that specifically decomposes IgA.

[25] The reagent kit for immunological assays according to

[24] , wherein the enzyme is a protease.

[26] The reagent kit for immunological assays according to

[25] , wherein the protease is a proteinase or a peptidase.

[27] The reagent kit for immunological assays according to any one of

[24] to

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

[28] The reagent kit for immunological assays according to any one of

[24] to

[27] , wherein the enzyme acts on IgA in the sample and decomposes the IgA into Fab fragments and Fc fragments.

[29] The reagent kit for immunological measurement according to any one of

[24] to

[28] , wherein the IgA is IgA1.

[30] A non-specific reaction inhibitor containing an enzyme that specifically degrades IgA. [Effects of the Invention]

[0012] According to the present invention, it is possible to provide an immunological measurement method that can suppress non-specific reactions caused by IgA contained in a measurement sample.

[0013] The immunological assay method of the present invention can suppress non-specific reactions that could not be suppressed even with conventional non-specific reaction inhibitors, thereby enabling accurate measurement of the target substance. DETAILED DESCRIPTION OF THE INVENTION

[0014] [Immunological measurement method] The immunoassay method of the present invention is a method for immunologically measuring a substance to be measured in a sample, characterized in that an antigen-antibody reaction is carried out at least once in the presence of an enzyme that specifically degrades IgA. In other words, the method is a method in which an enzyme that specifically degrades IgA is reacted with the sample as a non-specific reaction inhibitor, and the substance to be measured in the sample is immunologically measured using a specific binding partner in the presence of the enzyme.

[0015] One embodiment of the immunological assay method of the present invention is a non-specific reaction suppression method in which an antigen-antibody reaction is carried out at least once in the presence of an enzyme that specifically degrades IgA, and non-specific reactions in the reaction solution are suppressed during this reaction.

[0016] Immunological assay methods are broadly divided into homogeneous methods and heterogeneous methods.

[0017] The homogeneous method is a measurement method that specifically detects the binding reaction that occurs between the substance to be measured and its specific binding partner in a mixed solution (reaction solution) of sample and reagent solution without performing B / F (bound / unbound) separation.The heterogeneous method is a measurement method that performs B / F separation, washes out and removes excess components that were not involved in the binding reaction, and then allows the binding reaction to proceed to detect the substance to be measured.

[0018] The heterogeneous method has the drawback of requiring many steps and time for measurement due to the need for washing steps, but has the advantage of being relatively less susceptible to the effects of non-specific reaction substances.In contrast, the homogeneous method does not require a washing step and therefore has the drawback of being susceptible to the effects of non-specific reactions, but is simple with few steps and requires a short measurement time, making it a method that is widely sought after in the field of clinical diagnosis.

[0019] Homogeneous methods include immunoagglutination assays (IA), such as turbidimetric immunoassays (TIA) and immunochromatography (lateral flow and flow-through). TIA qualitatively or quantitatively detects a target substance in a sample based on the degree of agglutination of immune complexes formed by crosslinking of the target substance with a specific binding partner such as an antibody. Among these, latex turbidimetric immunoassays (LTIA), which use latex particles as an insoluble carrier to amplify the agglutination signal, are suitable for optical detection and can be easily automated, making them a versatile method applicable to a variety of test items.

[0020] Examples of heterogeneous methods include ELISA using well-shaped plates and chemiluminescence methods.

[0021] The present invention can be used for any of the above immunological measurement methods, but homogeneous methods, which are relatively susceptible to the influence of non-specific reactions, are preferred because they are expected to be more effective, and among homogeneous methods, latex immunoturbidimetry is most preferred.

[0022] [An enzyme that specifically degrades IgA] One embodiment of the present invention includes a method for immunoassay using an enzyme that specifically degrades IgA, or a non-specific reaction inhibitor containing the enzyme (hereinafter, an enzyme that specifically degrades IgA may be abbreviated as an IgA-specific degrading enzyme). The IgA-specific degrading enzyme may be any enzyme that can specifically degrade IgA, such as a protease. Examples of proteases include proteinases (endopeptidases), peptidases (exopeptidases), metalloproteases, serine proteases, and cysteine ​​proteases. In the present invention, the enzyme that degrades IgA is any enzyme that cleaves the amino acid sequence of the IgA constant region, including CHα1 to CHα3, preferably an enzyme that cleaves a portion of the amino acid sequence of the hinge region between CHα1 and CHα2 (e.g., VPSTPPTPSPST of human IgA: SEQ ID NO: 1), and more preferably an enzyme that can degrade IgA into Fab fragments and Fc fragments. The IgA-specific degrading enzyme only needs to have the function of specifically degrading IgA when it is brought into contact with a sample such as a specimen, and the enzyme may be inactivated by the time the subsequent antigen-antibody reaction is carried out.

[0023] As used herein, the terms "reacting with" an antibody and "recognizing" an antigen are used interchangeably, but are not limited to these examples and should be interpreted in the broadest sense. Whether an antibody "reacts" with an antigen can be confirmed by antigen-immobilized ELISA, competitive ELISA, sandwich ELISA, or the like, as well as by a method utilizing the principle of surface plasmon resonance (SPR). The SPR method can be performed using an apparatus, sensor, and reagents commercially available under the name Biacore (registered trademark).

[0024] In this specification, the terms "decompose" IgA by an enzyme, "react" with IgA by an enzyme, and "digest" IgA by an enzyme are used synonymously, but are not limited to these examples, and the meaning of "decompose" IgA by an enzyme should be interpreted in the broadest sense.

[0025] The IgA-specific enzyme of the present invention specifically degrades IgA. IgA-specific enzymes can degrade IgA more efficiently than immunoglobulins other than IgA, such as IgG and IgM. Therefore, even when the analyte is IgG or when the binding partner that specifically binds to the analyte is IgG, IgA-specific enzymes can be used without affecting the specific measurement of the analyte. Furthermore, IgA-specific enzymes can degrade IgA derived from organisms such as humans, mice, rats, rabbits, goats, ostriches, and pigs. When specifically degrading human IgA, it is preferable to use human IgA-specific enzymes. Human IgA, in particular, has two subclasses, IgA1 and IgA2. Human IgA-specific enzymes may specifically degrade human IgA1 and human IgA2, but it is more preferable that they specifically degrade human IgA1. IgA exists in both blood and secretory fluids, and is present mainly as a monomer in blood, whereas it exists as a dimer or multimer in secretory fluids. The IgA-specific degrading enzyme preferably degrades both IgA present in blood and IgA present in secretory fluids, but it may also degrade only one of them.

[0026] In the present invention, the IgA-specific degrading enzyme specifically degrades IgA means that, for example, after reacting IgG or IgA with the enzyme for 30 to 60 minutes at 37°C, the amount of IgA remaining is 50% or less, 40% or less, 30% or less, preferably 20% or less, and more preferably 10% or less, compared to the amount of IgG.

[0027] The IgA-specific enzyme used in the present invention may be either a naturally occurring enzyme or a recombinant enzyme produced by genetic engineering. Examples of naturally occurring enzymes include enzymes produced by various bacteria and fungi, such as those produced by bacteria that cause oral infections (Streptococcus sanguis, Streptococcus mitis, and Streptococcus oralis), bacteria that cause genital tract infections such as gonorrhea (Neisseria gonorrhoeae), and bacteria that cause meningitis (Haemophilus influenza, Neisseria meningitidis, and Streptococcus pneumoniae). (Patricia de Sousa-Pereira and Jenny M. Woof. (2019). IgA: Structure, Function, and Developability. Antibodies, 8, 57,) Preferably, IgASAP (manufactured by Genovis) or an amino acid variant of the enzyme is used. Alternatively, the enzyme may be a recombinant enzyme produced as a recombinant protein. By artificially introducing amino acid residue substitutions into recombinant enzymes and screening using various known assay systems, recombinant enzyme variants with desired enzymatic activity can also be produced (JP 2019-506866, EP 3148576). Therefore, in the present invention, various IgA-specific degrading enzymes can be used to perform methods for inhibiting nonspecific reactions in immunological assays and to produce nonspecific reaction inhibitors.

[0028] In the present invention, methods for introducing an enzyme that specifically degrades IgA into an antigen-antibody reaction system include using the enzyme as one of the components of an immunoassay reagent, or adding the enzyme to a sample dilution solution, a sample extract, a sample pretreatment solution, etc. For example, a sample dilution solution, a sample extract, or a sample pretreatment solution that already contains an IgA-specific degrading enzyme may be used, or the IgA-specific degrading enzyme may be added to a sample dilution solution, a sample extract, or a sample pretreatment solution. In this specification, the term "sample pretreatment solution" includes both sample dilution solutions and sample extracts.

[0029] For example, when the immunoassay reagent is a liquid reagent, the inside of the antigen-antibody reaction system refers to the liquid phase where the sample and the liquid immunoassay reagent are mixed and the antigen-antibody reaction takes place.

[0030] For example, in the case of the LTIA method, the sample may be mixed with an immunoassay reagent containing an IgA-specific degrading enzyme, or the sample may be mixed in advance with a specimen pretreatment solution containing an IgA-specific degrading enzyme and then mixed with the LTIA reagent.

[0031] In the case of the ELISA method, the sample may be mixed with the IgA-specific enzyme beforehand and then dropped onto the microplate, or the sample may be mixed with a solution containing the IgA-specific enzyme and the detection antibody before being dropped onto the microplate.

[0032] In the case of the chemiluminescence method, the sample may be mixed in advance with a specimen pretreatment solution containing an IgA-specific degrading enzyme and then mixed with the immunoassay reagent, or the immunoassay reagent (e.g., a solution containing a detection antibody or antigen and magnetic particles) may contain the IgA-specific degrading enzyme.

[0033] When an antigen-antibody reaction is carried out on a solid phase such as in immunochromatography, the term "inside the antigen-antibody reaction system" refers to the solid phase where the antigen-antibody reaction between a liquid sample and a binding partner takes place. In this case, the sample may be premixed with a specimen pretreatment solution containing an IgA-specific enzyme and then dropped onto the immunochromatographic test piece. Alternatively, the IgA-specific enzyme may be dried and held on a component such as a sample pad (sample supply site), and when the sample is dropped, the IgA-specific enzyme dissolves and develops the solid phase, thereby becoming present in the reaction system.

[0034] In the present invention, the concentration of the IgA-specific degrading enzyme may be any concentration that does not significantly affect the antigen-antibody reaction between the substance to be measured and its specific binding partner and that can exert the desired effect of inhibiting non-specific reactions, and can be appropriately determined by a person skilled in the art depending on the type of substance to be measured and the type of sample.

[0035] The concentration of the IgA-specific enzyme in the antigen-antibody reaction system varies depending on the reagent composition of the antigen-antibody reaction system. For example, when adding IgA-specific enzyme to a sample pretreatment solution, the enzyme can be added at a concentration of 1 to 1000 U, preferably 5 to 800 U, more preferably 10 to 500 U, even more preferably 15 to 300 U, and most preferably 20 to 200 U per 10 μL of sample. Furthermore, when adding IgA-specific enzyme to a sample pretreatment solution, the enzyme can be added at a concentration of 1 to 1000 U / μL, preferably 5 to 800 U / μL, more preferably 10 to 500 U / μL, even more preferably 15 to 300 U / μL, and most preferably 20 to 200 U / μL. Furthermore, 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.

[0036] In the present invention, IgA-specific enzymes may be used alone or in combination with other substances that have the effect of inhibiting non-specific reactions. Examples of other substances that have the effect of inhibiting non-specific reactions include anti-IgA antibodies, polymeric compounds, Heteroblock (OMEGA Biologicals), and modified antibodies in which the variable region of the L chain or H chain of a specific antibody has been partially or completely modified. It should be noted that other substances that have the effect of inhibiting non-specific reactions are not limited to these. The effect of inhibiting non-specific reactions is expected to be enhanced by using an IgA-specific enzyme in combination with other substances that have the effect of inhibiting non-specific reactions. Furthermore, when an anti-IgA antibody and an IgA-specific enzyme are used in combination, the anti-IgA antibody and the enzyme may be bound directly or indirectly.

[0037] When the IgA-specific degrading enzyme is to be contained in advance in the immunoassay reagent of the present invention, it is preferable that it be contained in advance in the assay reagent so as to have a concentration that is in the above-mentioned reaction system.

[0038] [Latex immunoturbidimetric assay (LTIA)] The LTIA method, which is one of the immunological measurement methods of the present invention, will now be described. Methods for measuring a substance to be measured by the LTIA method can be broadly divided into two types.

[0039] The first method involves reacting the substance to be measured with latex particles onto which a specific binding partner for the substance to be measured is immobilized to form a sandwich-type immune complex, and measuring the substance to be measured based on the degree of agglutination of the latex particles associated with the formation of the immune complex.

[0040] The second method involves adding proteins or the like to which multiple substances to be measured or their analogs (including fragments thereof) are immobilized to an immunoassay reagent, causing these to compete with the substances to be measured in a sample, thereby inhibiting the formation of immune complexes between the substances to be measured contained in the reagent and latex particles to which specific binding partners for the substances to be measured are immobilized, and measuring the substances to be measured (e.g., antigens) from the degree of inhibition of agglutination of the latex particles that accompanies the inhibition of immune complex formation.

[0041] The analyte and its specific binding partner may be any of proteins, peptides, sugar chains, lipids, glycoproteins, glycolipids, nucleic acids, low-molecular-weight compounds, and high-molecular-weight compounds, as long as they can specifically bind to the analyte, and any substance can be selected depending on the purpose. For example, if the analyte is an antigen, any antibody, such as a polyclonal antibody or a monoclonal antibody (including monoclonal antibodies produced by hybridomas, recombinant antibodies, and functional fragments of each antibody), can be selected as the specific binding partner for the analyte. If the analyte is an antibody, an antigen, such as a natural or recombinant antigen, can be selected as the specific binding partner for the analyte.

[0042] The present invention can be used in any of the above methods, and specific examples thereof include, but are not limited to, the following steps.

[0043] (1) A step of contacting a sample containing a substance to be measured with an IgA-specific degrading enzyme in a solution.

[0044] (2) After step (1), adding latex particles carrying a specific binding partner for the substance to be measured to the solution.

[0045] (3) After the step (2), a step of optically detecting the degree of aggregation of the latex particles in the solution.

[0046] Here, step (3) means "a step of measuring the agglutination reaction between the substance to be measured and the latex particles during or after step (2) without going through a washing or separation step."

[0047] The LTIA method measures the target substance by optically observing the degree of agglutination. Optical observation methods include measuring scattered light intensity, absorbance, or transmitted light intensity with an optical instrument (endpoint method, rate method, etc.). The change in absorbance measured in this way is used to calculate the concentration (quantitative value) of the target substance contained in the sample using a calibration curve obtained by measuring a standard substance with a known concentration of the target substance. Measurement of absorbance of transmitted light or scattered light may be performed using either a single wavelength measurement or a dual wavelength measurement (the difference or ratio between two wavelengths). Measurement wavelengths are generally selected from the range of 500 nm to 900 nm.

[0048] In the present invention, the measurement of the substance to be measured in the sample may be performed manually or using an apparatus such as a measuring device. The measuring device may be a general-purpose automatic analyzer or a dedicated measuring device (specialized machine). Furthermore, this measurement is preferably performed by a method involving multiple operational steps, such as a two-step method (two-reagent method).

[0049] [Latex particles carrying specific binding partners] The specific binding partner for the substance to be measured can be immobilized and supported on latex particles by known methods such as physical adsorption, chemical binding, or a combination of these. In the case of physical adsorption, the specific binding partner for the substance to be measured and latex particles can be mixed and contacted in a solution such as a buffer solution, or the specific binding partner for the substance to be measured dissolved in a buffer solution or the like can be contacted with a carrier, according to known methods. Furthermore, when using a chemical binding method, it can be carried out according to known methods described in, for example, "Clinical Pathology Extra Special Issue No. 53: Immunoassays for Clinical Tests - Techniques and Applications" edited by the Japanese Society of Clinical Pathology, Clinical Pathology Publishing Society, published in 1983; and "New Biochemical Experiment Course 1: Protein IV" edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, published in 1991, by mixing and contacting a specific binding partner for the substance to be measured and a carrier with a bivalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide, and then reacting the amino group, carboxyl group, thiol group, aldehyde group, hydroxyl group, or the like of the specific binding partner for the substance to be measured and the carrier with the bivalent cross-linking reagent.

[0050] The synthetic polymer constituting the latex particles is not particularly limited, and examples thereof include polystyrene, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, itaconic acid polymer, and styrene-hydrophilic carboxy monomer copolymer, such as styrene-methacrylic acid copolymer, styrene-acrylic acid copolymer, and styrene-itaconic acid copolymer. Among these, preferred are styrene-methacrylic acid copolymer, styrene-itaconic acid copolymer, and styrene and styrene-styrene sulfonate copolymer. Particularly preferred are styrene and styrene-(meth)acrylic acid copolymer.

[0051] It is preferable that the latex particles carry multiple types of specific binding partners for the analyte to form a sandwich. When the analyte has multiple antibody recognition sites, only one type of specific binding partner may be used. For example, when the specific binding partner is a monoclonal antibody, multiple monoclonal antibodies with different recognition sites are used. Furthermore, when the specific binding partner is a polyclonal antibody, the polyclonal antibody may be derived from one type of antiserum, or may be derived from multiple types of antisera. Furthermore, a combination of a monoclonal antibody and a polyclonal antibody may be used.

[0052] If treatment is required to suppress spontaneous aggregation of latex particles or nonspecific reactions, the surface of the latex particles may be treated by a known method, such as contacting and coating with a protein such as bovine serum albumin (BSA), casein, gelatin, egg albumin or a salt thereof, a surfactant, or skim milk powder, thereby blocking (masking) the carrier.

[0053] [Immunoassay reagents] The immunoassay method of the present invention can be carried out using an immunoassay reagent. The immunoassay reagent is characterized by containing the above-mentioned IgA-specific degrading enzyme in addition to the main components of the antigen-antibody reaction. The main components include a binding partner specific to the substance to be measured, as well as insoluble carriers such as immunoassay particles, immunochromatographic test strips, and microplates.

[0054] The immunoassay reagent of the present invention may contain buffers, proteins, peptides, amino acids, nucleic acids, lipids, phospholipids, sugars, glycoproteins, glycolipids, inorganic salts, polymeric compounds, surfactants, other nonspecific reaction inhibitors, preservatives, etc., to the extent that the nonspecific reaction inhibitory effect of the IgA-specific degrading enzyme is not impaired. As components for buffering and adjusting the pH, ionic strength, osmotic pressure, etc. of the sample, the reagent may contain, 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., Good's buffer, and sodium salts, potassium salts, calcium salts, etc. Furthermore, the reagent may contain polymers such as polyvinylpyrrolidone and phospholipid polymers as components for enhancing the agglutination formation of particles for immunoassay.

[0055] The concentration of the IgA-specific degrading enzyme in the constituent reagents may be such that it can be adjusted to the concentration in the antigen-antibody reaction system when the reagent and sample are mixed together at the time of measurement, and varies depending on the type of reagent.

[0056] [Immunoassay reagent kit] The immunoassay reagent kit of the present invention is characterized by including at least an IgA-specific enzyme in its components. Therefore, the reagent kit of the present invention includes the IgA-specific enzyme in one or more of the following components: a sample dilution solution, a sample pretreatment solution including a sample extract, and the like, in addition to the reagents involved in the antigen-antibody reaction that make up the kit. The kit also includes instructions for use and sample collection tools (such as a collection pipette, syringe, cotton swab, and filter). The reagent composition for each immunological measurement method will be explained below.

[0057] [Latex immunoturbidimetric assay] Examples of reagents (LTIA reagents) when the immunoassay is latex immunoturbidimetry are given below, but are not limited to these.

[0058] (1) Reagent 1 containing IgA-specific degrading enzyme (2) a second reagent containing latex particles carrying a specific binding partner for the substance to be measured;

[0059] The first reagent typically contains a buffer solution, and the concentration of the IgA-specific protease in the buffer solution may be adjusted to the preferred enzyme concentration when the reagent and sample are mixed together at the time of measurement, and this concentration varies depending on the type of reagent. The enzyme may be contained in the second reagent in addition to the first reagent.

[0060] In one example of the LTIA reagent of the present invention, the concentration of the IgA-specific protease contained in a typical 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 these concentrations.

[0061] When a sample pretreatment solution or the like is added to the sample before subjecting it to the LTIA method, the IgA-specific degrading enzyme may be contained in the sample pretreatment solution, and the enzyme concentration in the treatment 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 these concentrations.

[0062] (temperature) The reaction temperature of the IgA-specific enzyme with the sample may be any temperature at which the IgA-specific enzyme is not inactivated and can specifically decompose IgA, and is in the range of 0 to 60°C, preferably 10 to 50°C, and more preferably 20 to 40°C, but is not limited to these temperatures.

[0063] (Reaction time) The reaction time of the IgA-specific enzyme with the sample may be any reaction time that allows the IgA-specific enzyme to specifically and sufficiently decompose IgA, and is in the range of 30 seconds to 24 hours, preferably 1 minute to 120 minutes, but is not limited to this reaction time.

[0064] (pH) The pH of the solution containing the IgA-specific degrading enzyme may be any pH at which the IgA-specific degrading enzyme functions sufficiently, and is not limited to a pH range of 1 to 12, preferably 3 to 10, more preferably 5 to 9, and most preferably 6 to 8. Compared with known protease enzymes such as papain, pepsin, and trypsin, the enzymatic activity of the IgA-specific degrading enzyme is enhanced over a wider pH range.

[0065] (Immunological measurement particles) In addition to the above-mentioned latex particles, any known particles can be used as the particles for immunoassays in the present invention as long as they can carry a specific binding partner for the substance to be measured. For example, inorganic particles such as metal colloids, silica, carbon, and magnetic particles can also be used as the particles for immunoassays in the present invention.

[0066] The size of particles for immunological measurement can be appropriately selected from the range of 0.05 to 1 μm, taking into consideration the optical measurement method to be used (for example, turbidimetry, which measures transmitted light, or nephelometry, which measures scattered light), so as to obtain the desired measurement sensitivity, measurement range, etc. In optical measurements using automatic analyzers, an average particle size of 0.1 to 0.4 μm is generally used, but is not limited to this.

[0067] [ELISA method] ELISA is a method that utilizes a combination of various antigen-antibody reactions, ultimately incorporating an enzyme-labeled antigen or antibody into the reaction system to detect enzyme activity. To detect enzyme activity, a substrate whose absorption spectrum changes upon reaction is used, and various methods are available, depending on the combination of antigen-antibody reactions: direct, indirect, sandwich, and competitive.

[0068] The following is an example of a reagent for immunoassay when the immunoassay method of the present invention is a sandwich ELISA method.

[0069] (a) An insoluble carrier onto which an antibody that reacts with the substance to be measured is immobilized (b) An antibody labeled with a labeling substance and reacting with the substance to be measured

[0070] The insoluble carrier (a) is preferably a plate, and the labeling substance can be selected appropriately. The antibody immobilized on the insoluble carrier captures the analyte in a solution containing the sample and forms a complex on the insoluble carrier. The antibody labeled with a labeling substance binds to the captured analyte and forms a sandwich with the complex. The analyte in the sample can be measured by measuring the amount of the labeling substance using a method appropriate for the labeling substance. Specific methods, such as the method for immobilizing the antibody on the insoluble carrier and the method for binding the antibody to the labeling substance, can be methods well known to those skilled in the art and are not particularly limited.

[0071] In the ELISA method, the IgA-specific protease of the present invention can be present in the immune reaction system, for example, by adding it to a sample pretreatment solution or to a solution in which an antigen-antibody reaction is carried out.

[0072] [Immunochromatography method] The constitution of the immunoassay reagent (test strip constitution) when the immunoassay method of the present invention is an immunochromatography method will be described below. immunochromatographic test strips; When an antibody is used as the specific binding partner, the test piece is provided with, on a sheet-like insoluble carrier such as a porous membrane, in the direction in which the solution containing the sample is developed, "1. Sample supply site," "2. Site for holding a labeled antibody (labeled antibody holding site)," and "3. Site for immobilizing an antibody to capture the complex formed by the labeled antibody and the substance to be measured (capture antibody site)." In immunochromatography, when a predetermined amount of a sample containing the analyte is added to the sample supply site, the sample enters the labeled antibody holding site by capillary action, where the analyte binds to the labeled antibody to form a complex. The complex then spreads across the membrane and enters the capture antibody site, where it is captured by an antibody (capture antibody) immobilized on the membrane, forming a complex of capture antibody, analyte, and labeled antibody. The analyte can then be detected by detecting the label using any method (e.g., by detecting its agglutination in the case of a visible label such as gold colloid, or by a color reaction resulting from the addition of a substrate in the case of an enzyme). In the immunochromatography method, 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 containing it in a sample supply site or a labeled antibody holding site and drying and holding it there.

[0073] [Chemiluminescence method] After reacting the substance to be measured with magnetic particles bound to antigens or antibodies to form a complex, unreacted substances are removed by magnetism. A reagent containing a labeled antibody is then added, and after unreacted substances are removed by magnetism, a luminescent reagent is added and the amount of luminescence is measured. When an enzyme is used as the label, it is called a chemiluminescent enzyme immunoassay (CLEIA method). When a metal complex such as a ruthenium pyridine complex is used as the label and luminescence intensity is measured by an electrochemical reaction, it is called an electrochemiluminescence immunoassay (ECLIA method). When a chemiluminescent substance is used as the label, it is called a chemiluminescent immunoassay (CLIA method). The following is an example of an immunoassay reagent when the immunoassay method of the present invention is the CLEIA method. (a) Magnetic particles onto which antibodies (or antigens) that react with the substance to be measured are immobilized (b) An enzyme-labeled antibody (or antigen) that reacts with the substance to be measured (c) luminescent reagent The antibody immobilized on the magnetic particles captures the analyte in a solution containing the sample, forming a complex. The antibody labeled with an enzyme-labeled substance binds to the captured analyte and forms a sandwich with the complex. The analyte in the sample can be measured by reacting the enzyme-labeled substance with a luminescent reagent and measuring the amount of luminescence.

[0074] In the CLEIA method, the IgA-specific protease of the present invention can be present in the immune reaction system by, for example, adding it to a sample pretreatment solution or to a solution in which an antigen-antibody reaction is carried out.

[0075] [Specific binding partner] In the present invention, the specific binding partner for the analyte may 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, high molecular weight compounds, etc. Furthermore, there are no particular limitations on the molecular weight or origin (natural or synthetic), and examples include antibodies or antigens that can be used in immunological assays that utilize antigen-antibody reactions.

[0076] The antibody may be a polyclonal antibody or a monoclonal antibody, and is more preferably a monoclonal antibody.

[0077] <Sample> In the present invention, examples of samples containing a substance to be measured include human or animal blood, serum, plasma, culture supernatant, urine, cerebrospinal fluid, saliva, sweat, ascites, nasal discharge, feces, or cell or tissue extracts. Blood is the most preferred sample containing a substance to be measured. It is generally known that the reference value for IgA in blood is 110 to 410 mg / dL. The sample may also be referred to as a "specimen."

[0078] [Substances to be measured] The immunoassay reagent of the present invention can measure various substances contained in the sample. Examples of substances to be measured include proteins, peptides, amino acids, lipids, carbohydrates, glycoproteins, glycolipids, nucleic acids, and haptens, but there are no particular limitations as long as they are theoretically measurable. For example, C-reactive protein (CRP), lipoprotein(a) (Lp(a)), matrix metalloproteinase 3 (MMP3), antiphospholipid antibodies, type IV collagen, prostate specific Protein-specific antigen (PSA) (molecular weight: 34,000), brain natriuretic peptide (BNP) (molecular weight: 3,500), N-terminal pro-brain natriuretic peptide (NT-proBNP) (molecular weight: 8,500), insulin (molecular weight: 5,800), albumin, cystatin C, rheumatoid factor (RF), KL-6, procalcitonin (PCT) (molecular weight: 13,000), fibrin and fibrinogen degradation products (FDP), D-dimer, soluble fibrin (SF), thrombin-antithrombin III complex (TAT), transferrin, haptoglobin, α1-antitrypsin, α1-acid These include glycoprotein, α2-macroglobulin, hemopexin, antithrombin-III, α-fetoprotein, carcinoembryonic antigen (CEA), ferritin, hepatitis B virus envelope s antigen (HBs-Ag), anti-hepatitis B virus envelope s antibody (Anti-HBs), hepatitis B virus envelope e antigen (HBe-Ag), anti-hepatitis B virus envelope e antigen antibody (nti-HBe), anti-hepatitis B virus core antibody (Anti-HBc), severe acute respiratory syndrome virus (SARS), PAI-1 (molecular weight: 42,700), phenytoin, phenobarbital, carbamazepine, valproic acid, theophylline, thymus and activation-regulated chemokine (TARC) (molecular weight: 8,100), soluble interleukin-2 receptor (sIL-2R) (molecular weight: 45,000), and pulmonary surfactant protein D (SP-D). The molecular weight of the substance to be measured may be 50,000 or less, and is preferably 50,000 or less.

[0079] The substances to be measured in the present invention include anti-Treponema pallidum antibodies, anti-cyclic citrullinated peptide (CCP) antibodies, anti-Helicobacter pylori antibodies, and antibodies such as IgG and IgM against viruses such as hepatitis, measles, and leukemia. In the technology described in Patent Document 2, these antibodies may also be degraded by reducing agents. On the other hand, the IgA-specific enzyme of the present invention does not degrade these antibodies. Therefore, the IgA-specific enzyme is particularly useful for the technology described in Patent Document 2 when the substance to be measured is an antibody.

[0080] [Non-specific reaction inhibitors] In the present invention, "inhibiting nonspecific reactions" refers to acting on factors (also referred to as nonspecific factors, nonspecific causative substances, or nonspecific reaction substances) that cause the above-mentioned nonspecific reactions in biological samples, thereby suppressing the effects of reactions other than antigen-antibody reactions on the measurement. Therefore, in the present invention, whether a candidate substance as a nonspecific reaction inhibitor has the effect of inhibiting nonspecific reactions can be determined by comparing the measured values ​​(hereinafter referred to as control measured values) obtained using, for example, a measurement method with B / F separation (a method that includes a washing step and is less susceptible to the effects of nonspecific reaction substances (CLEIA method in the examples)) with and without the addition of the candidate substance and determining whether the measured values ​​are closer to the control measured values. In other words, if the measured values ​​obtained with the addition of the candidate substance in the target measurement method are closer to the control measured values ​​than the measured values ​​obtained without the addition of the candidate substance, it can be determined that the candidate substance has the effect of inhibiting nonspecific reactions in the measurement method, and that the candidate substance can be a nonspecific reaction inhibitor.

[0081] The nonspecific reaction inhibitor of the present invention targets both factors that cause a so-called positive measurement error, in which a measured substance is judged to have a higher content than its actual content, and factors that cause a so-called negative measurement error, in which a measured substance is judged to have a lower content than its actual value, due to some component contained in a biological sample. Among these, the inhibitor is particularly effective against nonspecific factors that cannot be suppressed by commercially available nonspecific reaction inhibitors such as HBR-1 and Heteroblock. The inhibitor is also effective against nonspecific factors that cause so-called deviation samples, which cause positive measurement errors that result in abnormally high measured values ​​and negative measurement errors that result in abnormally low measured values. In the present invention, various causative substances that cause nonspecific reactions can be cited, but it is preferable to degrade causative substances that have a structure similar to at least part, all, or part or all of IgA with an IgA-specific degrading enzyme. Through this degradation reaction, the nonspecific reaction inhibitor of the present invention can suppress nonspecific reactions caused by the causative substance.

[0082] The non-specific reaction inhibitor of the present invention may contain a substance capable of inhibiting a reaction caused by a non-specific factor derived from a sample, as determined above, and may contain at least an IgA-specific degrading enzyme as an active ingredient. The non-specific reaction inhibitor of the present invention may be configured such that the IgA-specific degrading enzyme is contained in the above-mentioned immunoassay reagent.

[0083] The non-specific reaction inhibitor of the present invention may contain a buffer, protein, peptide, amino acid, nucleic acid, lipid, phospholipid, saccharide, glycoprotein, glycolipid, inorganic salt, polymer compound, surfactant, other non-specific reaction inhibitors, preservatives, etc. within a range that does not interfere with the non-specific reaction inhibitory effect of the IgA-specific degrading enzyme. Other non-specific reaction inhibitors only need to have a non-specific reaction inhibitory effect, and examples include anti-IgA antibodies, polymer compounds, modified antibodies in which a part or all of the variable region of the L chain or H chain of a specific antibody is modified, but are not limited thereto. The combined use of an IgA-specific degrading enzyme and another substance having a non-specific reaction inhibitory effect is expected to enhance the non-specific reaction inhibitory effect. Furthermore, when an anti-IgA antibody and an IgA-specific degrading enzyme are used in combination, the anti-IgA antibody and the enzyme may be in a directly or indirectly bound state.

[0084] [Method for suppressing non-specific reaction] The method for suppressing non-specific reaction of the present invention is a method for suppressing non-specific reaction caused by a sample by performing an antigen-antibody reaction at least once in the presence of an IgA-specific degrading enzyme. Also, the method for suppressing non-specific reaction can be rephrased as a method for reducing measurement error.

[0085] Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following examples. [Examples]

[0086] [Suppression of non-specific reaction in LTIA method: Measurement of sIL-2R] The concentration of soluble interleukin 2 receptor (sIL-2R) contained in a sample (specimen) was measured by the LTIA method as follows. Human serum samples 1 to 3 from multiple individuals (3 individuals) were used as samples. Sample 1 (control sample) is a sample whose LTIA measurement values ​​are close to those of the chemiluminescent enzyme immunoassay (CLEIA) measurement values ​​(Reference Example 1). Samples 2 and 3 (deviation samples) are samples that exhibit a nonspecific reaction, and whose LTIA measurement values ​​deviate significantly from those of the CLEIA measurement values ​​in Reference Example 1. The IgA concentrations in each sample were 692 mg / dL for Sample 1, 387 mg / dL for Sample 2, and 247 mg / dL for Sample 3. The IgA concentrations of these samples were measured using the N-Assay TIA IgA-SH Nittobo reagent on a Hitachi 7180 automatic analyzer.

[0087] [Reference Example 1] Measurement by CLEIA method 1.Measurement method 1-1. Measurement reagents Determiner CL (registered trademark) IL-2R NX (Minaris Medical Co., Ltd.) 1-2.Sample Samples 1-3 1-3. Measurement procedure Measurement was performed using CL-JACK NX (registered trademark) (Minaris Medical Co., Ltd.) according to the instructions attached to the measurement reagent.

[0088] 2.Measurement results The measurement results are shown in Table 1. The CLEIA method shown in Reference Example 1 involves a B / F separation procedure and a washing step, making the CLEIA method a measurement method that is less susceptible to the influence of non-specific reactions derived from the sample.

[0089] [Comparative Example 1] Measurement by LTIA method: PBS buffer solution added to sample (no additives) 1.Measurement method 1-1. Measurement reagents The first and second reagents were prepared according to the method described in JP 2017-181377 A. 1-2. Sample pretreatment solution PBS buffer (pH 7.4) was used. 1-3. Sample PBS buffer solution, which is a sample pretreatment solution, was added to each of the samples (serum) 1 to 3 described in Reference Example 1 at a volume ratio of 5:1, and the samples were reacted at 37°C for 1 hour and then measured. 1-4. Measurement procedure The sample, first reagent, and second reagent were mixed for each specimen, and the sIL-2R concentration in the sample was measured using a Hitachi 7180 automatic analyzer. Specifically, 120 μL of the first reagent was added to 5.6 μL of sample and incubated at 37°C for 5 minutes. 40 μL of the second reagent was then added and stirred. The absorbance change associated with aggregate formation was measured over the next 5 minutes at a dominant wavelength of 570 nm and a sub-wavelength of 800 nm. The absorbance change was applied to a calibration curve obtained by measuring a standard substance of known concentration, and the measured value was calculated. Furthermore, the original solution equivalent value was calculated taking into account the dilution factor of the sample.

[0090] 2.Measurement results The measurement results converted to per 1 mL of serum before the addition of the sample pretreatment solution are shown in Table 1. The unit of the measurement results is U / mL.

[0091] [Example 1] Measurement by LTIA method: Addition of IgA-specific degrading enzyme to sample The measurement was carried out in the same manner as in Comparative Example 1, except that a PBS buffer solution containing 40 U / μL of IgASAP (manufactured by Genovis) was used as the sample pretreatment solution. The measurement results are shown in Table 1.

[0092] [Table 1]

[0093] Based on the results of Reference Example 1, Comparative Example 1 and Example 1, the inhibitory effect of the IgA-specific degrading enzyme on non-specific reactions was discussed. (1) The measurement results of the control sample (sample 1) were verified. For the control samples, the measured values ​​were generally equivalent in Reference Example 1, Comparative Example 1, and Example 1. These results demonstrate that the addition of IgASAP does not affect the measured values ​​of samples that do not exhibit nonspecific reactions. (2) The measurement results of the discrepant samples (samples 2 and 3) were verified. The measured value of Specimen 2 was 738 U / mL in Reference Example 1. In Comparative Example 1, it was 1419 U / mL, showing a deviation 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 approach that of Reference Example 1. A similar tendency was obtained for Specimen 3. The measured value of Specimen 3 was 630 U / mL in Reference Example 1. In Comparative Example 1, it was 1766 U / mL, showing a deviation 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 approach that of Reference Example 1. From the above, in the immunological measurement method, by using the IgA-specific degrading enzyme, the non-specific reaction derived from the sample could be suppressed. The discrepant specimens tested in this study were specimens for which sufficient non-specific reaction suppression effects could not be obtained only by using existing non-specific reaction inhibitors such as anti-IgA antibodies, and the non-specific reaction could be suppressed for the first time by the non-specific reaction suppression method of the present invention using the IgA-specific degrading enzyme.

[0094] [Experimental Example 2] [Suppression of non-specific reaction in the LTIA method: Measurement of sIL-2R] Similar to Example 1, the concentration of soluble interleukin 2 receptor (sIL-2R) contained in the sample (specimen) was measured by the LTIA method. The sample used was human serum specimen 2.

[0095] [Comparative Example 2] Measurement by the LTIA method: Addition of PBS buffer to the sample (without additives) 1. Measurement method 1-1. Measurement reagents According to the method described in JP-A-2017-181377, the first reagent and the second reagent were prepared. 1-2. Specimen pretreatment solution PBS buffer (pH 7.4) was used. 1-3. Sample The sample obtained by adding the PBS buffer, which was the specimen pretreatment solution, to the specimen (serum) 2 described in Reference Example 1 at a volume ratio of 4.5:1.5 and reacting at 37°C for 1 hour was measured. 1-4. Measurement procedure The specimen, first reagent, and second reagent were mixed, and the sIL-2R concentration in the sample was measured using a Hitachi 7180 automatic analyzer. Specifically, 120 μL of the first reagent was added to 5.6 μL of the sample and incubated at 37°C for 5 minutes. 40 μL of the second reagent was then added and stirred. The absorbance change associated with aggregate formation was measured over the next 5 minutes at a dominant wavelength of 570 nm and a sub-wavelength of 800 nm. The absorbance change was applied to a calibration curve obtained by measuring a standard substance of known concentration, and the measured value was calculated. Furthermore, the original solution equivalent value was calculated taking into account the dilution factor of the sample.

[0096] 2.Measurement results The measurement results converted to per 1 mL of serum before the addition of the sample pretreatment solution are shown in Table 2. The unit of the measurement results is U / mL.

[0097] [Reference Example 2] Measurement by LTIA method: Add a commercially available non-specific reaction inhibitor (Heteroblock) to the sample The measurement was carried out in the same manner as in Comparative Example 2, except that a PBS buffer solution containing 3.3 mg / mL Heteroblock (manufactured by OMEGA Biologicals) was used as the sample pretreatment solution. The measurement results are shown in Table 2.

[0098] [Example 2] Measurement by LTIA method: Addition of IgA-specific degrading enzyme to sample The measurement was carried out in the same manner as in Comparative Example 2, except that a PBS buffer solution containing 26.7 U / μL of IgASAP (manufactured by Genovis) was used as the sample pretreatment solution. The measurement results are shown in Table 2.

[0099] [Comparative Example 3] Measurement by LTIA method: Pepsin added to sample Measurement was carried out in the same manner as in Comparative Example 2, except that a PBS buffer solution containing 3.3 mg / mL Pepsin (manufactured by Roche, dissolved in 0.2 M citrate buffer, pH 3.0) was used as the sample pretreatment solution. The measurement results are shown in Table 2.

[0100] [Example 3] Measurement by LTIA method: Addition of IgA-specific decomposition enzyme and commercially available non-specific reaction inhibitor (Heteroblock) to the sample Measurements were performed in the same manner as in Comparative Example 2, except that a PBS buffer solution 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. The measurement results are shown in Table 2.

[0101] [Comparative Example 4] Measurement by LTIA method: Pepsin and a commercially available non-specific reaction inhibitor (Heteroblock) added to the sample Measurements were performed in the same manner as in Comparative Example 2, except that a PBS buffer solution containing 3.3 mg / mL Pepsin (Roche, dissolved in 0.2 M citrate buffer, pH 3.0) and 3.3 mg / mL Heteroblock (OMEGA Biologicals) was used as the sample pretreatment solution. The measurement results are shown in Table 2.

[0102] [Table 2]

[0103] Based on the results of Reference Example 1, Reference Example 2, Comparative Examples 2, 3, and 4, and Examples 2 and 3, the inhibitory effects of IgA-specific degrading enzyme, Pepsin, on non-specific reactions, and the combined effect with a commercially available non-specific reaction inhibitor (Heteroblock) were discussed.

[0104] (1) The effects of IgA-specific degrading enzyme and Pepsin were examined. The measured value of sample 2 in Reference Example 1 was 738 U / mL. The measured value in Comparative Example 2 (no additive) was 2092 U / mL, which deviated from that in Reference Example 1. The measured value in Example 2 (addition of IgA-specific degrading enzyme) was 977 U / mL, which tended to approach that in Reference Example 1. On the other hand, the measured value in Comparative Example 3 (addition of pepsin) was 2135 U / mL, which deviated from that in Reference Example 1 and was similar to that in Comparative Example 2 (no additive), and no inhibitory effect on non-specific reactions was observed.

[0105] (2) The effect of combining Pepsin with a commercially available nonspecific reaction inhibitor (Heteroblock) was examined. The measured values ​​for Sample 2 were 1180 U / mL in Reference Example 2 (heteroblock added) and 1271 U / mL in Comparative Example 4 (combined use of Pepsin and heteroblock), showing a tendency to deviate from those in Reference Example 1. This suggests that Pepsin is difficult to use in combination with other non-specific reaction inhibitors.

[0106] (3) The effect of combining IgA-specific degrading enzyme with a commercially available non-specific reaction inhibitor (Heteroblock) was examined. The measured values ​​for Sample 2 were 1180 U / mL in Reference Example 2 (with Heteroblock) and 977 U / mL in Example 2 (with IgA-specific enzyme), which were lower than those in Comparative Example 2 (without additives) and showed a significant improvement, but there was still room for improvement compared to the measured values ​​in Reference Example 1. On the other hand, the measured value in Example 3 (IgA-specific enzyme and Heteroblock combined) was 908 U / mL, which tended to approach that of Reference Example 1. This suggests that IgA-specific enzyme can be used in combination with other non-specific reaction inhibitors, and that the effect can be enhanced by further combination.

[0107] These results suggest that the use of IgA-specific enzymes in immunoassays successfully suppresses nonspecific reactions from samples. Furthermore, they also demonstrated a nonspecific reaction suppression effect on divergent samples for which the addition of pepsin was insufficient. IgA-specific enzymes can also be used in combination with commercially available nonspecific reaction inhibitors, and are significantly more useful than pepsin. The deviation samples used in this test were samples for which existing non-specific reaction inhibitors such as anti-IgA antibodies could not provide sufficient inhibitory effects, and non-specific reactions were successfully suppressed for the first time by the non-specific reaction inhibition method of the present invention using an IgA-specific degrading enzyme.

Claims

1. In an immunological measurement method for measuring a target substance in a sample, An immunological assay method in which an antigen-antibody reaction is carried out at least once in the presence of an enzyme that specifically degrades immunoglobulin A (IgA).

2. The immunoassay method according to claim 1 , wherein the enzyme is a protease.

3. The immunological assay method according to claim 2, wherein the protease is a proteinase or a peptidase.

4. 3. The immunological assay method according to claim 1, wherein the enzyme cleaves the amino acid sequence of the IgA constant region including CHα1 to CHα3.

5. 3. The immunological assay method according to claim 1, wherein the enzyme acts on IgA in the sample and decomposes 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. 3. The immunoassay method according to claim 1, wherein the immunoassay method is a latex immunoturbidimetric assay.

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

9. A reagent for immunoassay, which is used in an immunoassay method for measuring a substance to be measured in a sample, and which contains an enzyme that specifically degrades IgA.

10. The reagent for immunoassay according to claim 9, wherein the enzyme is a protease.

11. The reagent for immunoassay according to claim 10, wherein the protease is a proteinase or a peptidase.

12. The reagent for immunoassay according to claim 9 or 10, wherein the enzyme cleaves the amino acid sequence of the IgA constant region including CHα1 to CHα3.

13. The reagent for immunoassay according to claim 9 or 10, wherein the enzyme acts on IgA in the sample and decomposes the IgA into Fab fragments and Fc fragments.

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

15. The reagent for immunoassay according to claim 9 or 10, which is a reagent for use in latex immunoturbidimetry.

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

17. A sample pretreatment solution for immunological assays, comprising an enzyme that specifically degrades IgA.

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

19. 19. The sample pretreatment solution for immunological assays according to claim 18, wherein the protease is a proteinase or a peptidase.

20. 19. The sample pretreatment solution for immunological measurements according to claim 17, wherein the enzyme cleaves an amino acid sequence of an IgA constant region including CHα1 to CHα3.

21. 19. The specimen pretreatment solution for immunological measurements according to claim 17, wherein the enzyme acts on IgA in a sample and decomposes 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. 19. The sample pretreatment solution for immunological assay according to claim 17, wherein the sample pretreatment solution for immunological assay is a chemical solution for use in latex immunoturbidimetry.

24. A reagent kit for immunological measurement used in an immunological measurement method for measuring a substance to be measured in a sample, the reagent kit for immunological measurement comprising an enzyme that specifically degrades IgA.

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

26. The reagent kit for immunoassay according to claim 25, wherein the protease is a proteinase or a peptidase.

27. 26. The reagent kit for immunoassay according to claim 24, wherein the enzyme cleaves the amino acid sequence of the IgA constant region including CHα1 to CHα3.

28. 26. The reagent kit for immunoassay according to claim 24, wherein the enzyme acts on IgA in the sample and decomposes the IgA into Fab fragments and Fc fragments.

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

30. A non-specific reaction inhibitor containing an enzyme that specifically degrades IgA.

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