Immunological measurement method, reagent for immunological measurement, specimen pretreatment liquid for immunological measurement, reagent kit for immunological measurement, and non-specific reaction inhibitor

Abstract

Provided is an immunological measurement method for measuring a substance to be measured in a sample, wherein an antigen-antibody reaction is performed at least once in the presence of an enzyme specifically decomposing immunoglobulin M, abbreviated as IgM.

Description

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

[0001] The present invention relates to an immunoassay method, an immunoassay reagent, a sample pretreatment solution for immunoassay, a reagent kit for immunoassay, and a non-specific reaction inhibitor. This application claims priority to U.S. Provisional Application No. 63 / 534,585, filed on August 25, 2023, the contents of which are incorporated herein by reference.

[0002] Immunoassays, which utilize antigen-antibody reactions to measure target substances present in biological samples, are one example of a diagnostic drug measurement method. Because immunoassays utilize antigen-antibody reactions, they offer extremely high specificity. Various substances are present in biological samples, and measurement errors can occur due to nonspecific binding reactions or interference with specific antigen-antibody reactions caused by substances other than the target substance. This phenomenon is called a nonspecific reaction, and the substances that cause nonspecific reactions are called nonspecific factors. The existence of heterophilic antibodies and rheumatoid factors (RF) has been identified as nonspecific factors. Heterophilic antibodies are a collective term for human antibodies that are reactive with animal-derived antibodies, which are the primary reaction in immunoassays. Human anti-mouse immunoglobulin antibodies (HAMA) are a well-known example. Rheumatoid factors are glycoproteins that are frequently detected in patients with collagen diseases such as rheumatoid arthritis, chronic infections, and liver diseases. They share the characteristic of showing reactivity to animal-derived antibodies with HAMA, and both are known to be human immunoglobulin G or immunoglobulin M (hereinafter referred to as IgM) (Non-patent Documents 1 and 2).

[0003] Known techniques for suppressing nonspecific reactions in immunoassays include, for example, Patent Documents 1, 2, and 3. Patent Document 1 discloses a method for suppressing nonspecific reactions caused by RF by pretreating a sample with a sufficient amount of an animal-derived antibody 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 by adding a polyclonal antibody against natural IgM antibodies prepared from the same animal species as the antibody used in the assay. Patent Document 3 discloses a method for suppressing nonspecific reactions caused by interfering substances, such as rheumatoid factor, whose polypeptide chains are disulfide-bonded to each other, by using a reducing agent to cleave the disulfide bond and thereby decompose the interfering substance.

[0004] JP 07-012818 JP 11-287801 JP 13-255325

[0005] Clinical Chemistry; Vol. 23 Supplement 175a-1 to 175a-10 (1994) Immunoassay. Tetsuro Kubota, Kiyotaka Fujita, Eiji Hosoi, Michiko Kajiwara. Ishiyaku Publishing Co., Ltd. P. 199

[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 in practical use 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 polyclonal antibody against IgM type natural antibody shown in Patent Document 2 must be prepared from the same species of animal as the antibody used for measurement, and there are limitations on the preparation method and the antibody against the substance to be measured.

[0008] The reducing agent used in Patent Document 3 to suppress nonspecific reactions caused by interfering substances having a structure in which polypeptide chains are disulfide-bonded to each other may, depending on the type and concentration, cleave disulfide bonds in the antigen or antibody to be measured or in the antigen or antibody contained in the reagent components, making it less versatile. Furthermore, methods using reducing agents may also degrade IgG and IgA in addition to IgM. For this reason, this method could not be used, particularly when human IgG was the target substance to be measured.

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

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

[0011] [1] An immunoassay method for measuring a substance to be measured in a sample, comprising carrying out at least one antigen-antibody reaction in the presence of an enzyme that specifically degrades immunoglobulin M. [2] The immunoassay method according to [1], wherein the enzyme is a protease. [3] The immunoassay method according to [1] or [2], wherein the protease is a proteinase or a peptidase. [4] The immunoassay method according to any one of [1] to [3], wherein the enzyme is an enzyme that cleaves a part of the IgM constant region including CHμ1 to CHμ4. [5] The immunoassay method according to any one of [1] to [3], wherein the enzyme acts on IgM in the sample to cleave the IgM into F(ab') ２[6] The immunoassay method according to any one of [1] to [4], wherein the enzyme is an enzyme that degrades IgM into a β-amyloid fragment and an Fc fragment. [6] The immunoassay method according to any one of [1] to [5], wherein the immunoassay method is latex immunoturbidimetry. [7] The immunoassay method according to any one of [1] to [6], wherein the analyte is an antigen or an antibody. [8] A reagent used in an immunoassay method for measuring an analyte in a sample, the reagent comprising an enzyme that specifically degrades IgM. [9] The immunoassay reagent according to [8], wherein the enzyme is a protease.

[10] The immunoassay reagent according to [8] or [9], wherein the protease is a proteinase or peptidase.

[11] The immunoassay reagent according to any one of [8] to

[10] , wherein the enzyme is an enzyme that cleaves a part of the IgM constant region including CHμ1 to CHμ4.

[12] The enzyme acts on IgM in the sample to convert the IgM into F(ab') ２

[13] The immunoassay reagent according to any one of [8] to

[12] , wherein the immunoassay reagent is a reagent for use in latex immunoturbidimetry.

[14] The immunoassay reagent according to any one of [8] to

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

[15] A specimen pretreatment solution for immunoassays, comprising an enzyme that specifically degrades IgM.

[16] The specimen pretreatment solution for immunoassays according to

[15] , wherein the enzyme is a protease.

[17] The specimen pretreatment solution for immunoassays according to

[15] or

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

[18] The specimen pretreatment solution for immunoassays according to any one of

[15] to

[17] , wherein the enzyme is an enzyme that cleaves a part of the IgM constant region including CHμ1 to CHμ4.

[19] The enzyme acts on IgM in the sample to convert the IgM into F(ab') ２

[20] The specimen pretreatment solution for immunoassays according to any of

[15] to

[19] , wherein the specimen pretreatment solution for immunoassays is a chemical solution for use in latex immunoturbidimetry.

[21] A reagent kit for use in an immunoassay method for measuring a substance to be measured in a sample, the reagent kit for immunoassays comprising an enzyme that specifically degrades IgM.

[22] The reagent kit for immunoassays according to

[21] , wherein the enzyme is a protease.

[23] The reagent kit for immunoassays according to

[21] or

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

[24] The reagent kit for immunoassays according to

[22] or

[23] , wherein the enzyme is an enzyme that cleaves a part of the IgM constant region including CHμ1 to CHμ4.

[25] The enzyme acts on IgM in the sample to convert the IgM into F(ab') ２ The reagent kit for immunoassays according to any one of

[22] to

[24] , wherein the enzyme is an enzyme that degrades IgM into an IgM fragment and an Fc fragment.

[26] A non-specific reaction inhibitor comprising an enzyme that specifically degrades IgM.

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

[0014] 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 IgM. In other words, the method is a method in which an enzyme that specifically degrades IgM 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 aspect of the immunological assay method of the present invention is a method for suppressing nonspecific reactions, in which an antigen-antibody reaction is carried out at least once in the presence of an enzyme that specifically degrades IgM, and nonspecific 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 a specific binding partner in a mixed solution (reaction solution) of a sample and a reagent solution without performing B / F (bound / unbound) separation.The heterogeneous method is a measurement method that performs a B / F separation operation, washes out and removes excess components that were not involved in the binding reaction, and then allows the binding reaction to proceed and detects 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 is a method for qualitatively or quantitatively detecting an analyte in a sample based on the degree of agglutination of an immune complex formed by the crosslinking of the analyte (substance to be measured) with a specific binding partner such as an antibody. Among these, latex turbidimetric immunoassays (hereinafter sometimes referred to as 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 highly versatile method that is applied to a variety of test items.

[0020] Examples of the heterogeneous method include an ELISA method using a well-shaped plate and a chemiluminescence method.

[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] [Enzyme that specifically degrades IgM] One aspect of the present invention is a method for performing immunological assay using an enzyme that specifically degrades IgM, or a non-specific reaction inhibitor containing said enzyme (hereinafter, the enzyme that specifically degrades IgM may be abbreviated as IgM-specific degrading enzyme). The IgM-specific degrading enzyme may be any enzyme that can specifically degrade IgM, and examples thereof include proteases. Among proteases, proteinases (endopeptidases), peptidases (exopeptidases), etc. are examples. In addition, the enzyme that degrades IgM in the present invention is C Ｈ μ1 to C Ｈ It is preferably an enzyme that cleaves a part of the IgM constant region containing μ4 (for example, the constant region of human IgM from Ser124 to Tyr576: SEQ ID NO: 1), and F(ab') ２ It is preferable that the IgM-specific degrading enzyme can degrade IgM into an IgM fragment and an Fc fragment. The IgM-specific degrading enzyme only needs to have the function of specifically degrading IgM 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 antigen 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" an enzyme, "react" with an enzyme, and "digest" an enzyme are used synonymously, but are not limited to these examples, and the meaning of "decompose" an enzyme should be interpreted in the broadest sense.

[0025] The IgM-specific enzyme of the present invention specifically degrades IgM. The IgM-specific enzyme can degrade IgM more efficiently than immunoglobulins other than IgM, such as IgG and IgA. Therefore, even when the analyte is IgG or when the binding partner that specifically binds to the analyte is IgG, the IgM-specific enzyme can be used without affecting the specific measurement of the analyte. Furthermore, the IgM-specific enzyme can degrade IgM derived from organisms such as humans, mice, rats, rabbits, goats, ostriches, and pigs. When it is desired to specifically degrade human IgM, it is desirable to use a human IgM-specific enzyme.

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

[0027] The IgM-specific degrading enzyme used in the present invention may be either a naturally occurring enzyme or a recombinant enzyme produced by genetic recombination technology. Examples of naturally occurring enzymes include enzymes produced by various bacteria and fungi, such as enzymes produced by streptococci, Legionella bacteria, Candida, etc. Preferably, the immunoglobulin M-degrading enzyme of S. suis (Ig d) produced by streptococcus is used. Ｓｓｕｉｓ) (Seele et al., J. Bacteriol., 2013; 195, 930-940) or amino acid mutants of said enzyme, IgMBRAZOR (Genovis) or amino acid mutants of said enzyme. The above-mentioned enzymes may also be recombinant enzymes produced as recombinant proteins. By artificially introducing amino acid residue substitutions into recombinant enzymes and screening using various known assay systems, recombinant enzyme variants having the desired enzymatic activity can also be produced (JP 2019-506866, EP 3148576). Therefore, in the present invention, various IgM-specific degrading enzymes can be used to perform methods for inhibiting nonspecific reactions in immunological assays or to produce nonspecific reaction inhibitors.

[0028] In the present invention, methods for making an enzyme that specifically degrades IgM present in an antigen-antibody reaction system include using it as one of the reagent components of an immunoassay reagent, and adding it to a specimen dilution solution, specimen extract, specimen pretreatment solution, etc. For example, a specimen dilution solution, specimen extract, or specimen pretreatment solution that already contains an IgM-specific degrading enzyme may be used, or the IgM-specific degrading enzyme may be added to a specimen dilution solution, specimen extract, or specimen pretreatment solution. In this specification, specimen dilution solutions and specimen extracts are both referred to as specimen pretreatment solutions.

[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 immunological assay reagent containing an IgM-specific degrading enzyme, or the sample may be mixed in advance with a specimen pretreatment solution containing an IgM-specific degrading enzyme and then mixed with the LTIA assay reagent.

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

[0032] In the case of a chemiluminescent reagent, the sample may be mixed in advance with a specimen pretreatment solution containing an IgM-specific degrading enzyme and then mixed with the immunoassay reagent, or the immunoassay reagent (e.g., a solution containing a detection antibody or detection antigen and magnetic particles) may contain the IgM-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 is carried out. In this case, the sample may be premixed with a specimen pretreatment solution containing an IgM-specific enzyme and then dropped onto the immunochromatographic test piece, or the IgM-specific enzyme may be dried and held on a member such as a sample pad (sample supply site), and when the sample is dropped, the IgM-specific enzyme is dissolved and develops the solid phase, thereby becoming present in the reaction system.

[0034] In the present invention, the concentration of the IgM-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 IgM-specific enzyme in the antigen-antibody reaction system varies depending on the reagent composition of the antigen-antibody reaction system. For example, when adding an IgM-specific enzyme to a sample pretreatment solution, 1 to 1000 U of the enzyme can be added per 10 μL of sample, preferably 5 to 800 U, more preferably 10 to 500 U, even more preferably 20 to 300 U, and most preferably 50 to 200 U. When adding an IgM-specific enzyme to a sample pretreatment solution, the concentration of the enzyme in the pretreatment solution can be 1 to 1000 U / μL, preferably 5 to 800 U / μL, more preferably 10 to 500 U / μL, even more preferably 10 to 300 U / μL, and most preferably 20 to 200 U / μL. Furthermore, when an IgM-specific degrading enzyme is added to the specimen pretreatment solution, the mixing ratio of the specimen to the specimen 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, the IgM-specific degrading enzyme 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-IgM antibodies, polymeric compounds, and modified antibodies in which part or all of the variable region of the L chain or H chain of a specific antibody has been modified. It should be noted that other substances that have the effect of inhibiting non-specific reactions are not limited to those listed here. Furthermore, when an anti-IgM antibody and an IgM-specific degrading enzyme are used in combination, the anti-IgM antibody and the enzyme may be bound directly or indirectly.

[0037] When the IgM-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 Immunoturbidimetry (LTIA)] The LTIA method, which is one of the immunological measurement methods of the present invention, will be described below. Methods for measuring a target substance 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 can 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 IgM-specific degrading enzyme in a solution.

[0044] (2) After the step (1), a step of 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 target substance and the latex particles during or after step (2) without going through a washing / separation step."

[0047] The LTIA method can measure the target substance by optically observing the degree of agglutination. Optical observation methods include measuring scattered light intensity, absorbance, or transmitted light intensity using optical equipment (endpoint method, rate method, etc.). The measured values ​​of absorbance, etc. obtained by measuring a sample are compared with the measured values ​​of absorbance, etc. obtained by measuring a standard substance (a sample with a known concentration of the target substance), to calculate the concentration (quantitative value) of the target substance contained in the sample. Measurement of absorbance, etc., of transmitted light or scattered light may be performed using either a single wavelength or a dual wavelength (the difference or ratio between two wavelengths). The measurement wavelength is 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] Specific binding partners for the substance to be measured can be immobilized and carried 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 brought into contact with a carrier, according to known methods. Furthermore, when the chemical binding method is used, it can be carried out according to known methods described in, for example, "Clinical Pathology Extra Special Issue No. 53, Immunoassay for Clinical Testing - Techniques and Applications -" edited by the Japanese Society of Clinical Pathology, Clinical Pathology Publishing Society, published in 1983; "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, styrene-hydrophilic carboxy monomer copolymer such as styrene-methacrylic acid copolymer, styrene-acrylic acid copolymer, styrene-itaconic acid copolymer, vinylnaphthalene polymer, etc. Among these, preferred are styrene-methacrylic acid copolymer, styrene-itaconic acid copolymer, styrene and styrene-styrene sulfonate copolymer. 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. If the analyte has multiple antibody recognition sites, a single type of specific binding partner may be used. For example, if the specific binding partner is a monoclonal antibody, multiple monoclonal antibodies with different recognition sites are used. Furthermore, if 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 it is necessary to perform a treatment to suppress spontaneous aggregation of latex particles, non-specific reactions, etc., the surface of the latex particles may be treated by a known method, such as by contacting and coating the surface with a protein such as bovine serum albumin (BSA), casein, gelatin, egg albumin or a salt thereof, a surfactant, or skim milk powder, thereby performing a blocking treatment (masking treatment) of the carrier.

[0053] [Immunoassay Reagent] 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 IgM-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 IgM-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 IgM-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 IgM-specific enzyme in its kit configuration. Therefore, the reagent kit of the present invention includes the IgM-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. In addition to the above, the kit may also include an instruction manual and sample collection tools (collection pipettes, syringes, cotton swabs, filtration filters, etc.). The reagent configurations used for each immunoassay method are described below.

[0057] [Latex Immunoturbidimetry] Examples of reagents (LTIA reagents) when the immunological measurement method is latex immunoturbidimetry are given below, but are not limited to these.

[0058] (1) a first reagent containing an IgM-specific degrading enzyme; and (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 IgM-specific degrading enzyme in the buffer solution may be adjusted to the preferred enzyme concentration in the mixed state of the reagent and the sample at the time of measurement, and 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 IgM-specific degrading enzyme 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 a sample before subjecting the sample to the LTIA method, the IgM-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 IgM-specific degrading enzyme with the sample may be any temperature at which the IgM-specific degrading enzyme is not inactivated and can specifically degrade IgM, and is in the range of 0 to 50°C, preferably 10 to 40°C, and more preferably 20 to 40°C, but is not limited to these temperatures.

[0063] (Reaction Time) The reaction time of the IgM-specific degrading enzyme with the sample may be any reaction time that allows the IgM to be specifically and sufficiently degraded using the IgM-specific degrading enzyme, 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 IgM-specific degrading enzyme may be any pH at which the IgM-specific degrading enzyme functions sufficiently, 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 these pH values.

[0065] (Particles for immunological measurement) In addition to the above-mentioned latex particles, any known particles can be used as the particles for immunological measurement 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 immunological measurement in the present invention.

[0066] The size of the particles for immunological measurement can be appropriately selected from the range of 0.05 to 1 μm so as to obtain the desired measurement sensitivity, measurement range, etc., taking into consideration the optical measurement method to be used (for example, turbidimetry for measuring transmitted light, nephelometry for measuring scattered light, etc.). Note that, 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 for detecting enzyme activity by utilizing a combination of various antigen-antibody reactions, ultimately incorporating an enzyme-labeled antigen or antibody into the reaction system. To detect enzyme activity, a substrate whose absorption spectrum changes upon reaction is used, and methods include direct, indirect, sandwich, and competitive methods depending on the combination of antigen-antibody reactions.

[0068] The immunoassay reagents used when the immunoassay method of the present invention is a sandwich ELISA method are exemplified below.

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

[0070] The insoluble carrier (a) is preferably a plate, and the labeling substance can be appropriately selected and used. 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 IgM-specific degrading enzyme 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.

[0072] [Immunochromatography] The configuration of the immunoassay reagent (test strip configuration) when the immunoassay method of the present invention is an immunochromatography method will be described. Immunochromatographic test strip: When an antibody is used as a specific binding partner, this test strip comprises, in order of the direction of development of a sample-containing solution, a "1. sample supply site," a "2. site for holding a labeled antibody (labeled antibody retention site)," and a "3. site for immobilizing an antibody to capture a complex formed between the labeled antibody and the analyte (capture antibody site)" on a sheet-like insoluble carrier such as a porous membrane. In immunochromatography, when a predetermined amount of sample containing the analyte is added to the sample supply site, the sample enters the labeled antibody retention site by capillary action, where the analyte and the labeled antibody bind to form a complex. The complex develops through the membrane and enters the capture antibody site, where it is captured by an antibody (capture antibody) immobilized on the membrane, forming a capture antibody-analyte-labeled antibody complex. The substance to be measured can be detected by detecting the label by any method (for example, by detecting the agglutination image in the case of a visualized label such as gold colloid, or by a color reaction by adding a substrate in the case of an enzyme). In immunochromatography, the IgM-specific degrading enzyme of the present invention can be present in the reaction system by, for example, 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 therein.

[0073] [Chemiluminescence method] After reacting the substance to be measured with magnetic particles bound to an antigen or antibody to form a complex, unreacted substances are removed by magnetism. Furthermore, a reagent containing a labeled antibody is added, unreacted substances are removed by magnetism, and then a luminescent reagent is added to measure the amount of luminescence. 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).

[0074] Examples of immunoassay reagents when the immunoassay method of the present invention is the CLEIA method are as follows: (a) magnetic particles onto which an antibody (or antigen) that reacts with the substance to be measured is immobilized; (b) an enzyme-labeled antibody (or antigen) that reacts with the substance to be measured; and (c) a luminescent reagent. The antibody immobilized on the magnetic particles captures the substance to be measured in a solution containing a sample to form a complex. The antibody labeled with an enzyme-labeling substance binds to the captured substance to be measured, forming a sandwich with the complex. The substance to be measured in the sample can be measured by reacting the enzyme-labeling substance with the luminescent reagent and measuring the amount of luminescence.

[0075] In the CLEIA method, the IgM-specific degrading enzyme 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.

[0076] [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, and examples thereof include 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 the molecular weight or on the origin (natural or synthetic), and examples thereof include antibodies or antigens that can be used in immunological measurement methods that utilize antigen-antibody reactions.

[0077] The antibody may be a polyclonal antibody or a monoclonal antibody, and is more preferably a monoclonal antibody.

[0078] <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 known that the standard values ​​for IgM in blood are generally 33 to 190 mg / dl for men and 46 to 260 mg / dl for women. A sample may also be called a "specimen."

[0079] [Measurement Target Substance] The immunoassay reagent of the present invention can measure various substances contained in the sample. Measurement targets include proteins, peptides, amino acids, lipids, carbohydrates, glycoproteins, glycolipids, nucleic acids, and haptens, but are not particularly limited 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, and prostate specific antibodies. PSA, brain natriuretic peptide (BNP), insulin, albumin, cystatin C, rheumatoid factor (RF), KL-6, procalcitonin (PCT), fibrin and fibrinogen degradation products (FDP), D-dimer, soluble fibrin (SF), thrombin-antithrombin III complex (TAT), transferrin, haptoglobin, α1-antitrypsin, α1-acidoglycoprotein, 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 (anti-HBe), anti-hepatitis B virus core antibody (Anti-HBc), severe acute respiratory syndrome virus (SARS), PAI-1, phenytoin, phenobarbital, carbamazepine, valproic acid, theophylline, thymus and activation-regulated chemokine (TARC), soluble interleukin-2 receptor (sIL-2R), pulmonary surfactant protein D (SP-D), and the like.

[0080] The substances to be measured in the present invention include anti-Treponema (Treponema pallidum) antibodies, anti-cyclic citrullinated peptide (CCP) antibodies, anti-Helicobacter pylori antibodies, and antibodies such as IgG and IgA against viruses such as hepatitis, measles, and leukemia. In the technology described in Patent Document 3, these antibodies may also be degraded by reducing agents. On the other hand, the IgM-specific enzyme of the present invention does not degrade these antibodies. Therefore, the IgM-specific enzyme is particularly useful for the technology described in Patent Document 3 when the substance to be measured is an antibody.

[0081] [Non-specific reaction inhibitor] In the present invention, inhibiting a non-specific reaction means acting on factors (also referred to as non-specific factors, non-specific causative substances, or non-specific reaction substances) that cause the above-mentioned non-specific reaction in a biological sample, thereby suppressing the influence of reactions other than antigen-antibody reactions on the measurement. Therefore, in the present invention, whether a candidate substance as a non-specific reaction inhibitor has the effect of inhibiting a non-specific reaction can be determined by comparing the measured value (hereinafter referred to as the control method measured value) obtained by measurement using, for example, a measurement method with B / F separation (a method that has a washing step and is less susceptible to the influence of non-specific reaction substances (in the examples, the LBA method or the CLEIA method)) with and without the addition of the candidate substance and determining whether the measured value approaches the control method measured value. In other words, if the measured value obtained by adding the candidate substance in the target measurement method is closer to the control method measured value than the measured value obtained by not adding the candidate substance, it can be determined that the candidate substance has a non-specific reaction inhibitory effect in the measurement method, and that the candidate substance can be a non-specific reaction inhibitor.

[0082] The non-specific reaction inhibitor of the present invention targets both factors that cause a so-called positive measurement error, in which a substance to be measured is judged to have a higher content than its actual content, due to some component contained in a biological sample, and factors that cause a so-called negative measurement error, in which a substance to be measured is judged to have a lower content than its actual value. Among these, the inhibitor is particularly effective against non-specific factors that cannot be suppressed by commercially available non-specific reaction inhibitors such as HBR-1. The inhibitor is also effective against non-specific factors that cause positive measurement errors, in which measured values ​​become abnormally high, and negative measurement errors, in which measured values ​​become abnormally low, so-called deviation samples. In the present invention, various causative substances that cause non-specific reactions can be cited, but it is preferable to degrade causative substances having a structure similar to at least part, all, or part or all of immunoglobulin M (IgM) with an IgM-specific degrading enzyme. Through this degradation reaction, the non-specific reaction inhibitor of the present invention can suppress non-specific reactions derived from the causative substance.

[0083] 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 IgM-specific degrading enzyme as an active ingredient. The non-specific reaction inhibitor of the present invention may be configured such that the IgM-specific degrading enzyme is contained in the above-mentioned immunoassay reagent.

[0084] The non-specific reaction inhibitor of the present invention may contain buffers, proteins, peptides, amino acids, nucleic acids, lipids, phospholipids, sugars, glycoproteins, glycolipids, inorganic salts, polymeric compounds, surfactants, other non-specific reaction inhibitors, preservatives, etc., to the extent that the non-specific reaction inhibitory effect of the IgM-specific degrading enzyme is not impaired. Other non-specific reaction inhibitors may be any that have the effect of inhibiting non-specific reactions, and include, but are not limited to, anti-IgM antibodies, polymeric compounds, and modified antibodies in which part or all of the variable region of the L chain or H chain of a specific antibody has been modified. Furthermore, when an anti-IgM antibody and an IgM-specific degrading enzyme are used in combination, the anti-IgM antibody and the enzyme may be bound directly or indirectly to each other.

[0085] [Method for suppressing non-specific reactions] The method for suppressing non-specific reactions of the present invention is a method for suppressing non-specific reactions caused by a sample by performing an antigen-antibody reaction at least once in the presence of an IgM-specific protease. The method for suppressing non-specific reactions can also be referred to as a method for reducing measurement errors.

[0086] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0087] <Inhibition of Nonspecific Reactions in the LTIA Method: Measurement of PCT> The concentration of procalcitonin (PCT) contained in a sample (specimen) was measured by the LTIA method as follows. Human serum samples 1 and 2 from multiple individuals (2 individuals) were used as samples. Sample 1 (control sample) is a sample whose LTIA measurement value is close to the LBA (Liquid-Phase Binding Assay) measurement value (Reference Example 1). Sample 2 (deviation sample) is a sample that exhibits a nonspecific reaction, and whose LTIA measurement value deviates significantly from the LBA measurement value in Reference Example 1. The IgM concentration in each sample was 62 mg / dL in Sample 1 and 88 mg / dL in Sample 2.

[0088] Reference Example 1 Measurement by LBA Method 1. Measurement Method 1-1. Measurement Reagent: Mu-TAS Wako (registered trademark) PCT i50 (Fujifilm Wako Pure Chemical Industries, Ltd.) 1-2. Sample: Specimens 1 to 2 1-3. Measurement Procedure: Measurement was performed using Mu-TAS Wako (registered trademark) i50 (Fujifilm Wako Pure Chemical Industries, Ltd.) according to the package insert of the measurement reagent.

[0089] 2. Measurement Results The measurement results are shown in Table 1. The LBA method shown in Reference Example 1 involves B / F separation by isotachophoresis. Therefore, the LBA method is a measurement method that is less susceptible to the influence of nonspecific reactions derived from the sample.

[0090] Comparative Example 1 Measurement by LTIA method: PBS buffer solution added to sample (no additives) 1. Measurement method 1-1. Measurement reagents A first reagent and a second reagent were prepared according to the following method.

[0091] First Reagent A first reagent base solution was prepared having the following composition: 100 mM Bis-Tris-HCl (pH 6.5) 600 mM NaCl 0.2% BSA

[0092] Second reagent: 5 mM MOPS-NaOH (pH 7.0) Anti-human PCT monoclonal antibody-sensitized latex (two types) The anti-human PCT monoclonal antibody was obtained using a commercially available PCT antigen by a method known to those skilled in the art. The anti-human PCT monoclonal antibody-sensitized latex was prepared with reference to the method described in JP 2017-181377 A. That is, an anti-human PCT monoclonal antibody solution diluted to 0.36 mg / mL with an equivalent volume of 5 mM Tris-HCl (pH 8.5) was added to a 1.0% latex solution (5 mM Tris buffer solution (hereinafter referred to as Tris-HCl or simply Tris) (pH 8.5)) with an average particle size of 0.3 μm, and the mixture was stirred. Then, an equal volume of 5 mM Tris-HCl (pH 8.5) containing 0.5% BSA was added and the mixture was stirred to prepare an anti-human PCT monoclonal antibody-sensitized latex particle solution. This latex particle solution was diluted with 5 mM MOPS-NaOH (pH 7.0) so that the absorbance at 600 nm became approximately 5.5 OD, and this was used as the second reagent.

[0093] 1-2. Sample pretreatment solution PBS buffer solution (pH 7.4) was used.

[0094] 1-3. Samples PBS buffer solution, which is a sample pretreatment solution, was added to the specimens (serum) 1 and 2 described in Reference Example 1 at a volume ratio of 5:1, and the samples were reacted at 37°C for 30 minutes and measured.

[0095] 1-4. Measurement Procedure The sample, first reagent, and second reagent of each specimen were mixed, and the PCT concentration in the sample was measured using a Hitachi 3500 automatic analyzer. Specifically, 120 μL of the first reagent was added to 12 μL of sample and incubated at 37°C for 5 minutes. 40 μL of the second reagent was then added and stirred. The change in absorbance associated with agglutination formation was measured over the next 5 minutes at a dominant wavelength of 570 nm and a sub-wavelength of 800 nm. The amount of absorbance change was applied to a calibration curve obtained by measuring a standard substance of known concentration, and the measured value was calculated.

[0096] Example 1 Measurement by LTIA Method: Addition of IgM-Specific Degrading Enzyme to Sample 1. Measurement Method Measurement was performed in the same manner as in Comparative Example 1, except that a PBS buffer solution containing 40 U / μL of IgM BRAZOR (manufactured by Genovis), an IgM-specific degrading enzyme, was used as the sample pretreatment solution.

[0097] 2. Measurement Results The measurement results are shown in Table 1. The units of the values ​​of the measurement results are ng / mL.

[0098]

[0099] (1) The measurement results of control sample 1 (sample 1) were verified. For control sample 1, the measured values ​​in Reference Example 1, Example 1, and Comparative Example 1 were generally equivalent. These results demonstrated that the addition of IgM BRAZOR does not affect the measured values ​​of samples that do not exhibit nonspecific reactions.

[0100] (2) The measurement results of the deviation sample (sample 2) were verified. The measurement value of sample 2 in Reference Example 1 was 0.10 ng / mL. The measurement value in Comparative Example 1 was 1.68 ng / mL, which deviated from the measurement value in Reference Example 1. On the other hand, the measurement value in Example 1 was 0.02 ng / mL, which tended to approach that of Reference Example 1. From the above, it was found that the use of an IgM-specific degrading enzyme in an immunological measurement method was able to suppress non-specific reactions derived from the sample.

[0101] Comparative Example 2 Measurement by LTIA Method: Addition of Semi-Alkaline Protease to Sample Measurement was carried out in the same manner as in Example 1, except that proteinase from Aspergillus melleus, 5G (Sigma-Aldrich) diluted with PBS buffer to 0.4 mg / mL was used as the sample pretreatment solution. The measurement results are shown in Table 2.

[0102] Comparative Example 3 Measurement by LTIA Method: Addition of Semi-Alkaline Protease to Sample Measurement was carried out in the same manner as in Comparative Example 2, except that the enzyme concentration in the sample pretreatment solution was changed to 2.0 mg / mL. The measurement results are shown in Table 2.

[0103] Comparative Example 4 Measurement by LTIA Method: Addition of Proteinase K to Sample Measurement was performed in the same manner as in Comparative Example 2, except that the enzyme used was changed to Proteinase K, recombinant, PCR Grade, lyophilizate from Pichia pastoris (manufactured by Roche) and diluted with PBS buffer to a concentration of 0.4 mg / mL. The measurement results are shown in Table 2.

[0104] Comparative Example 5 Measurement by LTIA Method: Proteinase K Added to Sample Measurement was performed in the same manner as in Comparative Example 4, except that the enzyme concentration in the sample pretreatment solution was changed to 2.0 mg / mL. The measurement results are shown in Table 2. The numerical values ​​of the measurement results shown in Table 2 are in ng / mL.

[0105]

[0106] It can be seen that in control sample 1, comparative examples 2 to 5 showed significantly lower measured values ​​compared to example 1. The decrease was particularly pronounced when the enzyme concentration was high. It is believed that the use of a commercially available protease different from the IgM-specific protease degraded the PCT antigen in the control sample, resulting in a significant decrease in measured values. In deviation sample 2, comparative examples 2 and 4 showed higher measured values ​​compared to example 1. It is believed that the use of a commercially available protease different from the IgM-specific protease was unable to degrade the nonspecific causative substances in the deviation sample, resulting in the high deviation in measured values. From the above, it can be said that by using an IgM-specific protease in an immunological measurement method, it is possible to suppress nonspecific reactions derived from the sample without degrading the PCT antigen, which is the substance to be measured in the sample.

[0107] <Inhibition of Nonspecific Reactions in LTIA: Measurement of sIL-2R> The concentration of soluble interleukin-2 receptor (sIL-2R) contained in a sample (specimen) was measured by LTIA as follows. Human serum samples 4 to 6 from multiple individuals (3 individuals) were used as samples. Sample 4 (control sample) is a sample whose LTIA measurement value is close to the chemiluminescent enzyme immunoassay (CLEIA) measurement value (Reference Example 2). Samples 5 and 6 (deviation samples) are samples that exhibit a nonspecific reaction, and whose LTIA measurement value deviates significantly from the CLEIA measurement value in Reference Example 2. The IgM concentrations in each sample were 76 mg / dL in sample 4, 71 mg / dL in sample 5, and 532 mg / dL in sample 6.

[0108] Reference Example 2 Measurement by CLEIA Method 1. Measurement Method 1-1. Measurement Reagent Lumipulse Presto (registered trademark) IL-2R (Fujirebio Inc.) 1-2. Sample Specimens 4 to 6 1-3. Measurement Procedure Measurement was performed using Lumipulse (registered trademark)-L2400 (Fujirebio Inc.) according to the package insert of the measurement reagent.

[0109] 2. Measurement Results The measurement results are shown in Table 3. The CLEIA method shown in Reference Example 2 involves a B / F separation procedure and a washing step. Therefore, the CLEIA method is a measurement method that is less susceptible to the influence of nonspecific reactions derived from the sample.

[0110] Comparative Example 6: Measurement by LTIA Method: Addition of PBS Buffer 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. Samples: Samples (serum) 4 to 6 described in Reference Example 2 were treated with PBS buffer (sample pretreatment solution) at a volume ratio of 5:1, and the samples were incubated at 37°C for 1 hour and then measured. 1-4. Measurement Procedure: The sample, first reagent, and second reagent for each sample were mixed, and the sIL-2R concentration in the sample was measured using a Hitachi 7180 automated analyzer. Specifically, 120 μL of the first reagent was added to 5.6 μL of sample, and the mixture was incubated at 37°C for 5 minutes. 40 μL of the second reagent was then added and stirred. The change in absorbance due to the formation of aggregates was measured over the next 5 minutes at a dominant wavelength of 570 nm and a sub-wavelength of 800 nm. The change in absorbance was applied to a calibration curve obtained by measuring standard substances of known concentrations, and the measured value was calculated.

[0111] 2. Measurement Results The measurement results are shown in Table 3. The units of the values ​​of the measurement results are U / mL.

[0112] [Example 2] Measurement by LTIA method: Addition of IgM-specific degrading enzyme to sample Measurement was carried out in the same manner as in Comparative Example 6, except that a PBS buffer solution containing 40 U / μL of IgM BRAZOR (manufactured by Genovis) was used as the sample pretreatment solution. The measurement results are shown in Table 3.

[0113]

[0114] The effect of IgM-specific degrading enzymes on inhibiting nonspecific reactions was examined based on the results of Reference Example 2, Example 2, and Comparative Example 6. (1) The measurement results of the control sample (Sample 4) were verified. For the control sample, the measured values ​​of Reference Example 2, Example 2, and Comparative Example 6 were generally equivalent. These results demonstrated that the addition of IgMBRAZOR does not affect the measured values ​​of samples that do not exhibit nonspecific reactions. (2) The measurement results of the deviation samples (Samples 5 and 6) were verified. The measured value of Sample 5 in Reference Example 2 was 306 U / mL. The measured value of Comparative Example 6 was 556 U / mL, which deviated from the measured value of Reference Example 2. On the other hand, the measured value of Example 2 was 408 U / mL, tending to approach that of Reference Example 2. A similar trend was observed for Sample 6. The measured value of Sample 6 in Reference Example 2 was 591 U / mL. The measured value of Comparative Example 6 was 905 U / mL, which deviated from the measured value of Reference Example 2. On the other hand, the measurement result of Example 2 was 830 U / mL, which tended to approach that of Reference Example 2. From the above, it was found that the use of an IgM-specific degrading enzyme in an immunological measurement method was able to suppress non-specific reactions derived from the sample. The divergent samples used in this test were samples for which existing non-specific reaction inhibitors such as anti-IgM antibodies could not provide a sufficient inhibitory effect, and it was only by the non-specific reaction suppression method of the present invention using an IgM-specific degrading enzyme that non-specific reactions were able to be suppressed.

Claims

1. An immunoassay 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 M.

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. The immunological assay method according to claim 1 or 2, wherein the enzyme cleaves a part of the IgM constant region including CHμ1 to CHμ4.

5. The enzyme acts on IgM in the sample to convert the IgM into F(ab') ２ 3. The immunoassay method according to claim 1 or 2, wherein the enzyme is an enzyme that degrades the IgG1 fragment and the Fc fragment.

6. The immunoassay method according to claim 1 or 2, wherein the immunoassay method is a latex immunoturbidimetric assay.

7. The immunoassay method according to claim 6, wherein the substance to be measured is an antigen or an antibody.

8. A reagent for immunoassay used in an immunoassay method for measuring a substance to be measured in a sample, comprising an enzyme that specifically degrades IgM.

9. The reagent for an immunoassay according to claim 8, wherein the enzyme is a protease.

10. The reagent for an immunoassay according to claim 9, wherein the protease is a proteinase or a peptidase.

11. The reagent for immunoassay according to claim 8 or 9, wherein the enzyme cleaves a part of the IgM constant region including CHμ1 to CHμ4.

12. The enzyme acts on IgM in the sample to convert the IgM into F(ab') ２ The reagent for immunoassay according to claim 8 or 9, wherein the enzyme is an enzyme that degrades the IgG1 fragment and the Fc fragment.

13. The immunoassay reagent according to claim 8 or 9, which is a reagent for use in a latex immunoturbidimetric assay.

14. The immunoassay reagent according to claim 13, wherein the substance to be measured in the immunoassay method is an antigen or an antibody.

15. A sample pretreatment solution for immunological assays, containing an enzyme that specifically degrades IgM.

16. The sample pretreatment solution for immunological assays according to claim 15, wherein the enzyme is a protease.

17. The sample pretreatment solution for immunological assays according to claim 16, wherein the protease is a proteinase or a peptidase.

18. The sample pretreatment solution for immunological measurements according to claim 15 or 16, wherein the enzyme cleaves a part of the IgM constant region including CHμ1 to CHμ4.

19. The enzyme acts on IgM in the sample to convert the IgM into F(ab') ２ The sample pretreatment solution for immunological measurements according to claim 15 or 16, wherein the enzyme decomposes the IgG1 fragment and the Fc fragment.

20. The sample pretreatment liquid for immunological assay according to claim 15 or 16, which is a chemical liquid for use in latex immunoturbidimetry.

21. A reagent kit for immunoassay used in an immunoassay method for measuring a substance to be measured in a sample, the reagent kit comprising an enzyme that specifically degrades IgM.

22. The reagent kit for an immunoassay according to claim 21, wherein the enzyme is a protease.

23. The reagent kit for an immunoassay according to claim 22, wherein the protease is a proteinase or a peptidase.

24. The reagent kit for immunoassay according to claim 21 or 22, wherein the enzyme cleaves a part of the IgM constant region including CHμ1 to CHμ4.

25. The enzyme acts on IgM in the sample to convert the IgM into F(ab') ２ 23. The reagent kit for immunoassay according to claim 21 or 22, wherein the enzyme is an enzyme that degrades the IgG1 fragment and the Fc fragment.

26. A non-specific reaction inhibitor containing an enzyme that specifically degrades IgM.

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