Immunological assay methods, immunoassay reagents, sample pretreatment solutions for immunoassay, and immunoassay reagent kits; nonspecific reaction inhibitors.

Abstract

This invention provides an immunological assay method capable of suppressing nonspecific reactions caused by immunoglobulin M, abbreviated as IgM, contained in a sample for measurement, and reagents used in this immunological assay method. [Solution] The present invention provides an immunological assay method for measuring a target substance in a sample, comprising performing an antigen-antibody reaction at least once in the presence of an enzyme that specifically degrades IgM, and an immunological assay reagent used in the immunological assay method, comprising an enzyme that specifically degrades IgM.

Description

Technical Field

[0001] The present invention relates to an immunological measurement method, a reagent for immunological measurement, a specimen pretreatment solution for immunological measurement, a reagent kit for immunological measurement, and a non-specific reaction inhibitor. This application claims priority based on Provisional Application No. 63 / 534,585 filed in the United States on August 25, 2023, the content of which is incorporated herein by reference.

Background Art

[0002] As a measurement method in the field of diagnostic agents, there is an immunological measurement method for measuring a substance to be measured present in a biological sample by using an antigen-antibody reaction. Since the immunological measurement method utilizes an antigen-antibody reaction, it is a measurement method with very high specificity. Various substances are present in a biological sample, and a non-specific binding reaction occurs due to substances other than the substance to be measured, or the specific antigen-antibody reaction is hindered, resulting in a measurement error. Such a phenomenon is called a non-specific reaction, and substances that cause the non-specific reaction are called non-specific factors. As non-specific factors, the presence of heterophilic antibodies and rheumatoid factor (RF) has been clarified. Heterophilic antibodies are a general term for human antibodies that show reactivity with animal-derived antibodies responsible for the main reaction of immunological measurement methods, and human anti-mouse immunoglobulin antibody (HAMA) is known as a representative one. Rheumatoid factor is a glycoprotein that is frequently detected in patients with collagen diseases such as rheumatoid arthritis, chronic infectious diseases, and liver diseases, and has the common feature of showing reactivity with animal-derived antibodies, and its entity is known to be human immunoglobulin G or immunoglobulin M (hereinafter referred to as IgM) (Non-Patent Documents 1 and 2).

[0003] As a technique for suppressing non-specific reactions in immunological measurement methods, for example, Patent Documents 1, 2, and 3 are known. Patent Document 1 discloses a method for suppressing nonspecific reactions caused by RF by pre-treating a sample with a sufficient amount of animal-derived antibody that has the ability to bind to the antigen-binding site (Fab) of human rheumatoid factor. Examples of such animal-derived antibodies include anti-human immunoglobulin Fab antibody, anti-human IgG antibody (Fab-specific), anti-human IgA antibody (Fab-specific), and anti-human IgM antibody (Fab-specific). Patent Document 2 discloses a method for suppressing non-specific reactions by adding a polyclonal antibody against an IgM-type innate antibody prepared from the same animal species as the antibody used for measurement. Patent Document 3 discloses a method for suppressing nonspecific reactions caused by interfering substances having a structure in which polypeptide chains are bonded together by disulfide bonds, such as rheumatoid factor, by decomposing the interfering substance by cleaving the disulfide bonds using a reducing agent. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 07-012818 [Patent Document 2] Japanese Patent Application Publication No. 11-287801 [Patent Document 3] Japanese Patent Application Publication No. 13-255325 [Non-patent literature]

[0005] [Non-Patent Document 1] Clinical Chemistry; Vol. 23, Supplement 175a-1~175a-10 (1994) [Non-Patent Document 2] Immunology. Tetsuro Kubota, Kiyotaka Fujita, Eiji Hosoi, Michiko Kajiwara. Ishiyaku Publishers, Inc. P.199 [Overview of the project] [Problems that the invention aims to solve]

[0006] The anti-human IgM polyclonal antibodies, anti-human IgG polyclonal antibodies, and anti-human IgA polyclonal antibodies described in Patent Document 1 are currently being used in various reagents to suppress nonspecific reactions caused by natural antibodies and RF. However, it has become clear that this method cannot sufficiently suppress nonspecific reactions.

[0007] The polyclonal antibodies against IgM-type innate antibodies described in Patent Document 2 had to be prepared from the same species of animal as the antibody used for measurement, which limited the preparation method and the type of antibody used for the target substance.

[0008] The reducing agent described in Patent Document 3, used to suppress nonspecific reactions caused by interfering substances having a structure in which polypeptide chains are bonded by disulfide bonds, has low versatility because, depending on the type and concentration, it may cleave the disulfide bonds of the antigen or antibody being measured, or the antigen or antibody contained in the reagent component. Furthermore, methods using reducing agents may decompose not only IgM but also IgG and IgA. For this reason, this method could not be used, especially when human IgG was the target substance.

[0009] The object of the present invention is to provide an immunoassay method that can suppress nonspecific reactions caused by IgM contained in the measurement sample. [Means for solving the problem]

[0010] In order to solve the above problems, the inventors investigated the non-specific reaction suppression effects of various substances and found that non-specific reactions can be suppressed by performing an antigen-antibody reaction in the presence of an enzyme that specifically degrades IgM, thus completing the present invention. Specifically, the present invention has the following configuration.

[0011] [1] An immunoassay method for measuring a target substance in a sample, wherein an antigen-antibody reaction is performed at least once 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 portion of the IgM constant region including CHμ1 to CHμ4. [5] The immunoassay method according to any one of [1] to [4], wherein the enzyme acts on IgM in the sample and decomposes the IgM into F(ab')2 fragments and Fc fragments. [6] The immunological measurement method according to any one of [1] to [5], wherein the immunological measurement method is latex immunoturbidimetry. [7] The immunological measurement method according to any one of [1] to [6], wherein the substance to be measured is an antigen or an antibody. [8] An immunoassay reagent used in an immunoassay method for measuring a target substance in a sample, 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] An immunoassay reagent according to any one of [8] to

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

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

[11] , wherein the enzyme acts on IgM in the sample and decomposes the IgM into F(ab')2 fragments and Fc fragments.

[13] The immunoassay reagent is a reagent for use in latex immunoturbidimetry, as described in any of [8] to

[12] .

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

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

[15] A specimen pretreatment solution for immunological measurement, containing an enzyme that specifically decomposes IgM.

[16] The specimen pretreatment solution for immunological measurement according to

[15] , wherein the enzyme is a protease.

[17] The specimen pretreatment solution for immunological measurement according to

[15] or

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

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

[15] to

[18] , wherein the enzyme acts on IgM in the sample and decomposes the IgM into F(ab’)2 fragments and Fc fragments.

[20] The specimen pretreatment solution for immunological measurement according to any one of

[15] to

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

[21] A reagent kit for use in an immunological measurement method for measuring a substance to be measured in a sample, the reagent kit for immunological measurement containing an enzyme that specifically decomposes IgM.

[22] The reagent kit for immunological measurement according to

[21] , wherein the enzyme is a protease.

[23] The reagent kit for immunological measurement according to

[21] or

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

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

[22] to

[24] , wherein the enzyme acts on IgM in the sample and decomposes the IgM into F(ab’)2 fragments and Fc fragments.

[26] A non-specific reaction inhibitor containing an enzyme that specifically decomposes IgM.

Advantages of the Invention

[0012] According to the present invention, an immunological measurement method capable of suppressing non-specific reactions caused by IgM contained in a measurement sample can be provided.

[0013] In the immunological measurement method of the present invention, non-specific reactions that could not be suppressed even by using conventional non-specific reaction inhibitors can be suppressed. As a result, accurate measurement of the measurement target substance has become possible.

Mode for Carrying Out the Invention

[0014] [Immunological Measurement Method] The immunological measurement method of the present invention is a method for immunologically measuring a measurement target substance 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, as a non-specific reaction inhibitor, an enzyme that specifically degrades IgM is reacted with the sample, and in the presence of that enzyme, the measurement target substance in the sample is immunologically measured using a specific binding partner.

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

[0016] Immunological measurement methods are broadly classified into homogeneous methods and heterogeneous methods.

[0017] The homogeneous method is a measurement method that specifically detects a binding reaction occurring between a measurement target substance and a specific binding partner in a mixed solution (reaction solution) of a sample and a reagent solution without performing B / F (bound / free) separation. The heterogeneous method is a measurement method that performs a B / F separation operation, washes and removes excess components that did not participate in the binding reaction, and then progresses the binding reaction to detect the measurement target substance.

[0018] The heterogeneous method has the drawback of being time-consuming due to the washing step, but it has the advantage of being relatively less susceptible to the influence of non-specific reactants. In contrast, the homogeneous method does not involve a washing step, and therefore is susceptible to the influence of non-specific reactions, but it is simpler, has fewer steps, and requires less time for measurement, making it a widely sought-after method in the field of clinical diagnosis.

[0019] Homogeneous methods include immunoagglutination assays (IA), such as immunoturbidimetric assay (TIA) and immunochromatography (lateral flow and flow-through). TIA is a method for qualitatively or quantitatively detecting analytes in a sample based on the degree of aggregation of immune complexes formed by the crosslinking of analytes (target substances) by specific binding partners such as antibodies. Among these, latex immunoturbidimetric assay (LTIA), which uses latex particles as an insoluble carrier to amplify the agglutination signal, is suitable for optical detection and is easily automated, making it a highly versatile measurement method applied to various test items.

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

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

[0022] [Enzymes that specifically break down IgM] One aspect of the present invention is a method for performing immunological measurements 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 can be any enzyme that can specifically degrade IgM, for example, a protease. Among proteases, examples include proteinases (endopeptidases) and peptidases (exopeptidases). Furthermore, in the present invention, the enzyme that degrades IgM is C H μ1 to C H The enzyme is preferably one that cleaves a portion of the constant region of IgM containing μ4 (for example, the constant region of human IgM from Ser124 to Tyr576: SEQ ID NO: 1), and more preferably one that can degrade it into F(ab')2 and Fc fragments. The IgM-specific degrading enzyme only needs to have the function of specifically degrading IgM when it comes into contact with a sample such as a specimen; it may be inactivated by the time the subsequent antigen-antibody reaction is carried out.

[0023] In this specification, the terms "reacting" with an antigen and "recognizing" an antigen are used synonymously, but are not limited to these examples and should be interpreted in the broadest sense. Whether or not an antibody "reacts" with an antigen can be confirmed by antigen-immobilized ELISA, competitive ELISA, sandwich ELISA, etc., as well as by methods utilizing the principle of surface plasmon resonance (SPR method). The SPR method can be performed using equipment, sensors, and reagents commercially available under the name Biacore®.

[0024] In this specification, the terms "an enzyme breaks down" IgM, "an enzyme reacts" with IgM, and "an enzyme digests" IgM are used synonymously, but are not limited to these examples, and the meaning of "an enzyme breaks down" IgM should be interpreted in the broadest sense.

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

[0026] In the present invention, the IgM-specific degrading enzyme specifically degrades IgM, which means that, for example, after reacting IgG or IgM with the enzyme at 37°C for 30 to 60 minutes, the amount of remaining IgM is reduced to 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 engineering technology. Examples of naturally occurring enzymes include those produced by various bacteria and fungi, such as those produced by Streptococcus, Legionella, and Candida. Preferably, the immunoglobulin M-degrading enzyme of S. suis (Ig d) produced by Streptococcus suis is used. SsuisExamples include (Seele et al, J. Bacteriol., 2013; 195, 930-940) or amino acid variants of the enzyme, IgMBRAZOR (Genovis) or amino acid variants of the enzyme. Furthermore, the above-mentioned enzyme may be a recombinant enzyme produced as a recombinant protein. By artificially introducing amino acid residue substitutions into a recombinant enzyme and performing screening using various known assay systems, recombinant enzyme variants with desired enzyme activity can also be produced (JP 2019-506866, EP3148576). Therefore, in the present invention, various IgM-specific degrading enzymes can be used to implement methods for suppressing nonspecific reactions in immunological assays or to produce nonspecific reaction inhibitors.

[0028] In the present invention, methods for incorporating an enzyme that specifically degrades IgM into the antigen-antibody reaction system include using it as one of the reagent components of an immunoassay reagent, or adding it to a sample diluent, sample extract, or sample pretreatment solution. For example, a sample diluent, sample extract, or sample pretreatment solution containing an IgM-specific degrading enzyme beforehand may be used, or the IgM-specific degrading enzyme may be added to the sample diluent, sample extract, or sample pretreatment solution. In this specification, the sample pretreatment solution includes the sample diluent and sample extract.

[0029] The term "antigen-antibody reaction system" refers to the liquid phase in which the sample and the liquid immunoassay reagent are mixed and the antigen-antibody reaction takes place, for example, when the immunoassay reagent is a liquid reagent.

[0030] For example, in the case of the LTIA method, the sample may be mixed with an immunoassay reagent containing an IgM-specific degrading enzyme, or the sample may be pre-mixed with a sample pretreatment solution containing an IgM-specific degrading enzyme before being mixed with the LTIA assay reagent.

[0031] In the ELISA method, the sample may be pre-mixed with an IgM-specific degrading enzyme before being added to the microplate, or the sample may be mixed with a solution containing an IgM-specific degrading enzyme and a detection antibody before being added to the microplate.

[0032] In the case of chemiluminescence reagents, the sample may be mixed beforehand with a sample pretreatment solution containing an IgM-specific degrading enzyme before being mixed with the immunoassay reagent, or the immunoassay reagent (for example, a solution containing a detection antibody or detection antigen and magnetic particles) may contain an IgM-specific degrading enzyme.

[0033] In the case of antigen-antibody reactions carried out on a solid phase, such as by immunochromatography, the antigen-antibody reaction system refers to the solid phase in which the antigen-antibody reaction between the liquid sample and its binding partner takes place. In this case, the sample may be pre-mixed with a sample pretreatment solution containing an IgM-specific degrading enzyme before being dropped onto the immunochromatographic test piece, or the IgM-specific degrading enzyme may be kept dry on a component such as a sample pad (sample supply area), and when the sample is dropped, the IgM-specific degrading enzyme may dissolve and develop on the solid phase, becoming present in the reaction system.

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

[0035] The concentration of IgM-specific degrading enzyme in the antigen-antibody reaction system varies depending on the reagent composition of the antigen-antibody reaction system. For example, when adding IgM-specific degrading enzyme to a sample pretreatment solution, 1 to 1000 U, 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 can be added per 10 μL of sample. Alternatively, when adding IgM-specific degrading enzyme to a sample pretreatment solution, the concentration of 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 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, the IgM-specific degrading enzyme may be used alone or in combination with other substances that have a nonspecific reaction inhibitory effect. Examples of other substances that have a nonspecific reaction inhibitory effect include anti-IgM antibodies, high molecular weight 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. However, other substances that have a nonspecific reaction inhibitory effect are not limited to those listed herein. Furthermore, when using an anti-IgM antibody and an IgM-specific degrading enzyme in combination, the anti-IgM antibody and the enzyme may be directly or indirectly bound.

[0037] When the immunoassay reagent of the present invention contains IgM-specific degrading enzymes in advance, it is preferable to include them in the reagent beforehand at a concentration that matches the concentration within the reaction system described above.

[0038] [Latex immunoturbidimetric imaging (LTIA)] This section describes the LTIA method, one of the immunological measurement methods of the present invention. Methods for measuring target substances using the LTIA method can be broadly classified into two categories.

[0039] The first method involves reacting latex particles immobilized with a specific binding partner for the substance to be measured with the substance to be measured to form a sandwich-type immune complex, and measuring the substance to be measured from the degree of aggregation of the latex particles accompanying the formation of the immune complex.

[0040] The second method involves adding proteins or the like immobilized with multiple target substances or their analogues (including fragments thereof) to an immunoassay reagent, and causing competition between these and the target substances in the sample to inhibit the formation of an immune complex between the target substances contained in the reagent and latex particles immobilized with a specific binding partner for the target substances. The target substance (e.g., an antigen) is then measured from the degree of inhibition of aggregation of the latex particles due to the inhibition of immune complex formation.

[0041] The substance to be measured and its specific binding partner can be any protein, peptide, glycan, lipid, glycoprotein, glycolipid, nucleic acid, small molecule compound, or large molecule compound, as long as they can specifically bind to the substance to be measured. Any substance can be selected depending on the purpose. For example, if the substance to be measured is an antigen, any antibody can be selected as the specific binding partner, such as a polyclonal antibody or a monoclonal antibody (including monoclonal antibodies produced from hybridomas, recombinant antibodies, and functional fragments of each antibody). If the substance to be measured is an antibody, antigens such as natural and recombinant antigens can be selected as the specific binding partner.

[0042] The present invention can be used in any of the above methods, and specifically, the following steps are exemplified, but are not limited thereto.

[0043] (1) A step of bringing a sample containing the substance to be measured into contact with an IgM-specific degrading enzyme in solution.

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

[0045] (3) A step after step (2) in which the degree of aggregation of the latex particles in the solution is optically detected.

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

[0047] The LTIA method allows for the measurement of a target substance by optically observing the degree of aggregation that occurs. Methods for optical observation include measuring scattered light intensity, absorbance, or transmitted light intensity using optical instruments (endpoint method, rate method, etc.). The absorbance and other measurements obtained from measuring the sample are compared with the absorbance and other measurements obtained from 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. The measurement of absorbance, etc., of transmitted or scattered light may be performed using a single wavelength or a two-wavelength measurement (difference or ratio between two wavelengths). The measurement wavelength is generally selected from 500 nm to 900 nm.

[0048] In the present invention, the measurement of the target substance in a sample may be performed manually or using a measuring device. The measuring device may be a general-purpose automated analyzer or a dedicated measuring device (dedicated machine). Furthermore, it is preferable to carry out this measurement by a method consisting of multiple operating 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 thereof. In the case of physical adsorption, this can be done by mixing and contacting the specific binding partner for the substance to be measured and the latex particles in a solution such as a buffer, or by contacting the specific binding partner for the substance to be measured dissolved in a buffer, etc., with a carrier, according to known methods. Furthermore, when performing the test using a chemical binding method, it can be carried out by following known methods described in publications such as "Special Issue No. 53 of Clinical Pathology: Immunoassays for Clinical Tests - Techniques and Applications," edited by the Japanese Society of Clinical Pathology, published by the Clinical Pathology Publication Association in 1983; and "New Biochemistry Experiment Course 1: Protein IV," edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin in 1991. This involves mixing and contacting a specific binding partner for the target substance and a carrier with a divalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide, and then reacting the amino group, carboxyl group, thiol group, aldehyde group, or hydroxyl group of the specific binding partner for the target substance and the carrier with the aforementioned divalent crosslinking reagent.

[0050] There are no particular limitations on the synthetic polymers that constitute the latex particles, but examples include polystyrene, styrene-styrene sulfonate copolymers, methacrylic acid polymers, acrylic acid polymers, itaconic acid polymers, and styrene-hydrophilic carboxymonomer copolymers: for example, styrene-methacrylic acid copolymers, styrene-acrylic acid copolymers, styrene-itaconic acid copolymers, vinylnaphthalene polymers, etc. Among these, styrene-methacrylic acid copolymers, styrene-itaconic acid copolymers, and styrene and styrene-styrene sulfonate copolymers are preferred. Preferably, styrene and styrene-(meth)acrylic acid copolymers are preferred.

[0051] The specific binding partners for the target substance supported by the latex particles are preferably of multiple types in order to form a sandwich. If the target substance has multiple antibody recognition sites, one specific binding partner may suffice. For example, if the specific binding partner is a monoclonal antibody, multiple monoclonal antibodies with different recognition sites may be used. Also, for example, if the specific binding partner is a polyclonal antibody, it may be a polyclonal antibody derived from one type of antiserum, or from multiple types of antiserum. Furthermore, a combination of monoclonal antibodies and polyclonal antibodies may be used.

[0052] If it is necessary to perform treatment to suppress spontaneous aggregation of latex particles or nonspecific reactions, the surface of the latex particles may be treated by known methods, such as contacting and coating it with proteins such as bovine serum albumin (BSA), casein, gelatin, ovalbumin or its salts, surfactants, or skim milk powder, to perform a blocking treatment (masking treatment) of the carrier.

[0053] [Reagents for immunoassays] The immunological measurement method of the present invention can be performed using an immunological assay reagent. The immunological assay reagent is characterized by containing, in addition to the main component of the antigen-antibody reaction, the aforementioned IgM-specific degrading enzyme. The main component may be a binding partner specific to the substance to be measured, or it may be an insoluble carrier such as immunological assay particles, immunochromatographic test pieces, or 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, polymer compounds, surfactants, other nonspecific reaction inhibitors, preservatives, etc., to the extent that they do not interfere with the nonspecific reaction inhibitory effect of IgM-specific degrading enzymes. Components for buffering and adjusting the pH, ionic strength, osmotic pressure, etc., of the sample may include, for example, buffers such as acetic acid, citric acid, phosphoric acid, Tris, glycine, boric acid, carbonic acid, phthalic acid, succinic acid, maleic acid, imidazole, and Good's buffer, as well as their sodium, potassium, and calcium salts. Furthermore, polymers such as polyvinylpyrrolidone and phospholipid polymers may be included as components to enhance the aggregation of immunoassay particles.

[0055] The concentration of the IgM-specific degrading enzyme in the reagent should be such that it can be adjusted to the concentration within the antigen-antibody reaction system in the mixed state of the reagent and sample at the time of measurement, and this concentration will vary depending on the reagent type.

[0056] [Immunological assay reagent kit] The immunological assay reagent kit of the present invention is characterized by containing at least an IgM-specific degrading enzyme in its kit composition. Therefore, the reagent kit of the present invention contains an IgM-specific degrading enzyme in one or more of the following components, including reagents involved in the antigen-antibody reaction that constitute the kit, as well as a sample pretreatment solution including a sample diluent and a sample extract. In addition to the above, the kit composition may include instructions for use and sample collection tools (collection pipette, syringe, cotton swab, filter, etc.). The reagent configurations used for each immunological measurement method are described below.

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

[0058] (1) First reagent containing IgM-specific degrading enzyme (2) 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 should be such that it can be adjusted to the preferred enzyme concentration in the mixed state of the reagent and sample at the time of measurement, and this concentration varies depending on the reagent type. The enzyme may also be contained in the second reagent in addition to the first reagent.

[0060] In an example of the LTIA reagent of the present invention, the concentration of IgM-specific degrading enzyme contained in the first reagent is typically 1 to 1000 μg / mL, preferably 5 to 500 μg / mL, more preferably 10 to 100 μg / mL, and even more preferably 20 to 50 μg / mL, but is not limited to this concentration.

[0061] When a sample pretreatment solution is added to the sample before subjecting it to the LTIA method, the IgM-specific degrading enzyme may be included in the sample pretreatment solution. 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 this concentration.

[0062] (temperature) The reaction temperature of the IgM-specific degrading enzyme with the sample should be such that the IgM-specific degrading enzyme is not inactivated and IgM can be specifically degraded. This temperature range is 0 to 50°C, preferably 10 to 40°C, and more preferably 20 to 40°C, but is not limited to this range.

[0063] (Reaction time) The reaction time for the IgM-specific degrading enzyme with the sample should be any time that allows for the specific and sufficient degradation of IgM using the IgM-specific degrading enzyme, and should be in the range of 30 seconds to 24 hours, preferably 1 minute to 120 minutes, but not limited to this reaction time.

[0064] (pH) The pH of the solution containing the IgM-specific degrading enzyme should be any pH at which the IgM-specific degrading enzyme functions effectively, and is in the range of pH 1 to 12, preferably 3 to 10, more preferably 5 to 9, and most preferably 6 to 8, but is not limited to this pH.

[0065] (Immunological measurement particles) In addition to the latex particles described above, the immunoassay particles used in this invention can be any known particles that can support 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 immunoassay particles in this invention.

[0066] The particle size for immunological measurements can be appropriately selected from the range of 0.05 to 1 μm, taking into consideration the optical measurement method used (e.g., turbidimetry for measuring transmitted light, turbidimetry for measuring scattered light, etc.), in order to obtain the desired measurement sensitivity and measurement range. While an average particle diameter of 0.1 to 0.4 μm is commonly used in optical measurements using automated analyzers, the size is not limited to this range.

[0067] [ELISA method] ELISA is a method that utilizes various combinations of antigen-antibody reactions, ultimately incorporating enzyme-labeled antigens or antibodies into the reaction system to detect enzyme activity. For enzyme activity detection, substrates whose absorption spectra change during the reaction are used, and various methods such as direct, indirect, sandwich, and competitive methods are employed depending on the combination of antigen-antibody reactions.

[0068] Examples of immunoassay reagents used when the immunoassay method of the present invention is the sandwich ELISA method are provided.

[0069] (a) Insoluble carrier on which an antibody that reacts with the substance to be measured is immobilized. (b) An antibody labeled with a labeling substance that reacts with the substance to be measured.

[0070] (a) A plate is preferred as the insoluble carrier, and the labeling substance can be selected and used as appropriate. The antibody immobilized on the insoluble carrier captures the target substance in the solution containing the sample and forms a complex on the insoluble carrier. The antibody labeled with the labeling substance binds to the captured target substance and forms a sandwich with the aforementioned complex. The target substance in the sample can be measured by measuring the amount of the labeling substance using a method appropriate to 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 any method well known to those skilled in the art without particular limitation.

[0071] In the ELISA method, the IgM-specific degrading enzyme of the present invention can be made present in the immune response system by, for example, adding it to a sample pretreatment solution or adding it to a solution used in an antigen-antibody reaction.

[0072] [Immunochromatography Method] The composition of the reagent for immunoassay (test piece composition) when the immunoassay method of the present invention is immunochromatography will be described. Immunochromatographic specimen; When an antibody is used as the specific binding partner, the test piece is a sheet-like insoluble carrier such as a porous membrane, and in the order of the direction in which the solution containing the sample is deployed, it comprises: 1. a sample supply site, 2. a site for holding the labeled antibody (labeled antibody holding site), and 3. a 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, a predetermined amount of a sample containing at least the above-described test piece and the substance to be measured is added to the sample supply site. The sample enters the labeled antibody retention site by capillary action, and the substance to be measured and the labeled antibody bind to form a complex. When the membrane is deployed and the complex enters the capture antibody site, it is captured by the antibody immobilized on the membrane (capture antibody), forming a capture antibody-substance-labeled antibody complex. The substance to be measured can then be detected by detecting the label using any method (for example, by detecting the agglutination image in the case of a visible 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, for example, by adding it to a sample pretreatment solution or by incorporating it into the sample supply site or labeled antibody retention site and maintaining it in a dry state.

[0073] [Chemiluminescence] This method involves reacting magnetic particles bound to an antigen or antibody with the substance to be measured to form a complex, then removing unreacted substances by magnetism. Further, a reagent containing a labeled antibody is added, unreacted substances are removed by magnetism, and finally, a luminescent reagent is added to measure the amount of luminescence. When an enzyme is used for labeling, it is called chemiluminescent enzyme immunoassay (CLEIA). When a metal complex such as a ruthenium pyridine complex is used for labeling and the luminescence intensity is measured by an electrochemical reaction, it is called electrochemiluminescence immunoassay (ECLIA). When a chemiluminescent substance is used for labeling, it is called chemiluminescent immunoassay (CLIA).

[0074] Examples of immunoassay reagents used when the immunoassay method of the present invention is the CLEIA method are provided. (a) Magnetic particles immobilized with antibodies (or antigens) that react with the substance to be measured. (b) An enzyme-labeled antibody (or antigen) that reacts with the substance to be measured. (c) Luminescent reagent Antibodies immobilized on magnetic particles capture the target substance in the solution containing the sample and form a complex. Antibodies labeled with an enzyme-labeled substance bind to the captured target substance and form a sandwich with the aforementioned complex. By reacting the enzyme-labeled substance with a luminescent reagent and measuring the amount of light emitted, the target substance in the sample can be measured.

[0075] In the CLEIA method, the IgM-specific degrading enzyme of the present invention can be made present in the immune response system by, for example, adding it to a sample pretreatment solution or adding it to a solution used in an antigen-antibody reaction.

[0076] [Specific binding partners] In the present invention, the specific binding partner for the substance to be measured can be any substance that can specifically bind to the substance to be measured, and examples 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 restrictions on molecular weight or origin (natural or synthetic), but examples include antibodies or antigens that can be used in immunoassay methods utilizing antigen-antibody reactions.

[0077] The antibody may be a polyclonal antibody or a monoclonal antibody. More preferably, it is a monoclonal antibody.

[0078] <Sample> In this invention, examples of samples containing the substance to be measured include human or animal blood, serum, plasma, culture supernatant, urine, cerebrospinal fluid, saliva, sweat, ascites, nasal secretions, feces, or cell or tissue extracts. Blood is the most preferred sample containing the substance to be measured. Generally, the reference range for IgM in blood is known to be 33-190 mg / dl for men and 46-260 mg / dl for women. Samples are sometimes referred to as "specimens".

[0079] [Substances to be measured] The immunoassay reagent of the present invention can measure various substances contained in the sample. Examples of target substances include proteins, peptides, amino acids, lipids, carbohydrates, glycoproteins, glycolipids, nucleic acids, and haptens, but there are no particular limitations as long as the substance is theoretically measurable. For example, C-reactive protein (CRP), lipoprotein(a) (Lp(a)), matrix metalloproteinase 3 (MMP3), antiphospholipid antibodies, type IV collagen, and prostate-specific Antigen (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-acid glycoprotein, α2-macroglobulin, hemopexin, antithrombin- Examples include III, α-fetoprotein, carcinoembryonic antigen (CEA), ferritin, hepatitis B virus coat s antigen (HBs-Ag), anti-hepatitis B virus coat s antibody (Anti-HBs), hepatitis B virus coat e antigen (HBe-Ag), anti-hepatitis B virus coat e antigen antibody (nti-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), and pulmonary surfactant protein D (SP-D).

[0080] The substances to be measured in this 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 were also at risk of being degraded by the reducing agent. On the other hand, the IgM-specific degrading enzyme in this invention does not degrade these antibodies. For this reason, the IgM-specific degrading enzyme is particularly useful compared to 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, suppressing nonspecific reactions means acting on factors that cause the above-mentioned nonspecific reactions in a biological sample (also called nonspecific factors, nonspecific causative substances, or nonspecific reaction substances) to suppress the influence 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 an effect of suppressing nonspecific reactions can be determined by comparing the measured value (hereinafter referred to as the control method measurement) obtained when measured using a measurement method with B / F separation (a method that has a washing step and is less susceptible to the influence of nonspecific reaction substances (LBA method or CLEIA method in the examples)) with and without the candidate substance added, and seeing whether the result approaches the control method measurement value. That is, in the target measurement method, if the measured value when the candidate substance is added is closer to the control method measurement value than the measured value when the candidate substance is not added, then it can be determined that the candidate substance has a nonspecific reaction inhibitory effect in the measurement method and can be used as a nonspecific reaction inhibitor.

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

[0083] The nonspecific reaction inhibitor of the present invention only needs to contain a substance capable of suppressing reactions caused by nonspecific factors derived from the sample, as determined above, and contains at least an IgM-specific degrading enzyme as an active ingredient. The nonspecific reaction inhibitor of the present invention can adopt a configuration in which the above-mentioned immunoassay reagent includes an IgM-specific degrading enzyme as the configuration of the nonspecific reaction inhibitor.

[0084] The nonspecific reaction inhibitor of the present invention may contain buffers, proteins, peptides, amino acids, nucleic acids, lipids, phospholipids, sugars, glycoproteins, glycolipids, inorganic salts, polymer compounds, surfactants, other nonspecific reaction inhibitors, preservatives, etc., to the extent that they do not interfere with the nonspecific reaction inhibitory effect of the IgM-specific degrading enzyme. Other nonspecific reaction inhibitors only need to have a nonspecific reaction inhibitory effect, and include, but are not limited to, anti-IgM antibodies, polymer 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 using an anti-IgM antibody and an IgM-specific degrading enzyme in combination, the anti-IgM antibody and the enzyme may be directly or indirectly bound.

[0085] [Method for Suppressing Nonspecific Reaction] The method for suppressing nonspecific reaction of the present invention is a method for suppressing nonspecific reaction caused by a sample by performing an antigen-antibody reaction at least once in the presence of an IgM-specific degrading enzyme. Also, the method for suppressing nonspecific reaction can be rephrased as a method for reducing measurement error.

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

Example

[0087] [Suppression of Nonspecific Reaction in LTIA Method: Measurement of PCT] The concentration of procalcitonin (PCT) contained in a sample (specimen) was measured by the LTIA method as follows. As samples, a plurality of (two) human serum specimens 1 to 2 were used. Specimen 1 (control specimen) is a sample whose measured value by the LTIA method shows a value close to the measured value by the LBA method (Liquid-phase Binding Assay) (Reference Example 1). Specimen 2 (deviating specimen) is a sample that exhibits a nonspecific reaction and whose measured value by the LTIA method greatly deviates from the measured value of Reference Example 1 by the LBA method. The concentration of IgM in each specimen was 62 mg / dL for Specimen 1 and 88 mg / dL for Specimen 2.

[0088] [Reference Example 1] Measurement by LBA Method 1. Measurement Method 1-1. Measurement Reagent Mutuswa Co., Ltd. (registered trademark) PCT·i50 (FUJIFILM Wako Pure Chemical Corporation) 1-2. Sample Specimens 1 to 2 1-3. Measurement Procedure Measurement was performed using Mutuswa Co., Ltd. (registered trademark) i50 (FUJIFILM Wako Pure Chemical Corporation) according to the attached document of the measurement reagent.

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

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

[0091] Reagent 1 A first reagent base solution with the following composition was prepared. • 100 mM Bis-Tris-HCl (pH 6.5) 600mM NaCl 0.2% BSA

[0092] Reagent 2 5 mM MOPS-NaOH (pH 7.0) • Anti-human PCT monoclonal antibody-sensitized latex (2 types) The anti-human PCT monoclonal antibody was obtained using a commercially available PCT antigen by a method well known to those skilled in the art. The latex sensitized with the anti-human PCT monoclonal antibody was prepared with reference to the method described in Japanese Patent Publication No. 2017-181377. Specifically, a 1.0% latex solution with an average particle size of 0.3 μm (5 mM Tris buffer (hereinafter referred to as Tris-HCl or simply Tris) (pH 8.5)) was mixed with an equivalent volume of 5 mM Tris-HCl (pH 8.5) to which an anti-human PCT monoclonal antibody solution diluted to 0.36 mg / mL was added and the mixture was stirred. Then, an equivalent volume of 0.5% BSA-containing 5 mM Tris-HCl (pH 8.5) 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 was approximately 5.5 OD to prepare the second reagent.

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

[0094] 1-3. Samples Samples (serum) 1-2 described in Reference Example 1 were prepared by adding PBS buffer, a sample pretreatment solution, in a 5:1 volume ratio and reacting them at 37°C for 30 minutes.

[0095] 1-4. Measurement Procedure The PCT concentration in each sample was measured using a Hitachi 3500 automated analyzer after mixing the sample, reagent 1, and reagent 2. Specifically, 120 μL of reagent 1 was added to 12 μL of the sample and incubated at 37°C for 5 minutes. Then, 40 μL of reagent 2 was added and stirred. The change in absorbance due to aggregation formation was measured over the following 5 minutes at a primary wavelength of 570 nm and a secondary wavelength of 800 nm. The measured values ​​were calculated by applying the amount of absorbance change to a calibration curve obtained by measuring standard substances of known concentration.

[0096] [Example 1] Measurement by LTIA method: IgM-specific degrading enzyme added to the sample. 1.Measurement method The measurement was performed in the same manner as in Comparative Example 1, except that a PBS buffer containing 40 U / μL of IgM BRAZOR (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 unit of the numerical values ​​in the measurement results is ng / mL.

[0098] [Table 1]

[0099] (1) The measurement results of control sample 1 (sample 1) were verified. In control sample 1, the measured values ​​for reference example 1, example 1, and comparative example 1 were generally equivalent. These results demonstrate that the addition of IgMBRAZOR does not affect the measured values ​​of samples that do not exhibit a nonspecific reaction.

[0100] (2) The measurement results of the discrepant sample (sample 2) were verified. The measured value of sample 2 was 0.10 ng / mL for Reference Example 1. Comparative Example 1 was 1.68 ng / mL, which deviated from the measured value of Reference Example 1. On the other hand, the measured value of Example 1 was 0.02 ng / mL, showing a tendency to approach that of Reference Example 1. Based on the above, it was possible to suppress non-specific reactions originating from the sample by using IgM-specific degrading enzymes in immunoassay methods.

[0101] [Comparative Example 2] Measurement by LTIA method: Semi-alkaline protease added to the sample The measurement was performed using the same method as in Example 1, except that proteinase from Aspergillus melleus, 5G (Sigma-Aldrich) diluted in 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: Semi-alkaline protease added to the sample The measurement was performed using the same method 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: Proteinase K added to the sample The measurement was performed using the same method as in Comparative Example 2, except that the enzyme used was changed to Proteinase K, recombinant, PCR Grade, Lyophilizate from Pichia pastoris (Roche), and the concentration was diluted to 0.4 mg / mL in PBS buffer. The measurement results are shown in Table 2.

[0104] [Comparative Example 5] Measurement by LTIA method: Proteinase K added to the sample The measurement was performed using the same method 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 units of the measurement results listed in Table 2 are ng / mL.

[0105]

Table 2

[0106] In the control specimen 1, it can be seen that the measured values of Comparative Examples 2 to 5 were significantly lower than those of Example 1. Particularly, it was significantly decreased when the enzyme concentration was high. Since a commercially available protease different from the IgM-specific degrading enzyme decomposed the PCT antigen in the control specimen, it is considered that the measured value was significantly decreased. In the deviation specimen 2, the measured values of Comparative Examples 2 and 4 were higher than those of Example 1. Even when a commercially available protease different from the IgM-specific degrading enzyme was used, the non-specific causative substances in the deviation specimen could not be decomposed, and it is considered that the measured value deviated to a high value. From the above, in the immunological measurement method, it can be said that by using the IgM-specific degrading enzyme, the non-specific reaction derived from the sample could be suppressed without decomposing the PCT antigen which is the substance to be measured in the sample.

[0107] <Suppression of non-specific reaction in LTIA method: Measurement of sIL-2R> The concentration of soluble interleukin 2 receptor (sIL-2R) contained in the sample (specimen) was measured by the LTIA method as follows. As samples, sera specimens 4 to 6 from multiple (3) humans were used. Specimen 4 (control specimen) is a sample whose measured value by the LTIA method shows a value close to the measured value by the chemiluminescent enzyme immunoassay (CLEIA method) (Reference Example 2). Specimens 5 and 6 (deviation specimens) exhibit non-specific reactions, and the measured values by the LTIA method deviate significantly from the measured value of Reference Example 2 by the CLEIA method. The concentration of IgM in each specimen was 76 mg / dL in Specimen 4, 71 mg / dL in Specimen 5, and 532 mg / dL in Specimen 6.

[0108] [Reference Example 2] Measurement by CLEIA method 1. Measurement method 1-1. Measurement reagent Lumipulse Presto (registered trademark) IL-2R (FUJIREbio Inc.) <Object 1-2. Sample Samples 4-6 1-3. Measurement Procedure Measurements were performed using Lumipulse®-L2400 (Fujirebio Inc.) in accordance with the instructions provided for the aforementioned 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 operation and a washing step. Therefore, the CLEIA method is a measurement method that is less susceptible to the influence of nonspecific reactions originating from the sample.

[0110] [Comparative Example 6] Measurement by LTIA method: PBS buffer added to the sample (no additives). 1.Measurement method 1-1. Measurement Reagents The first reagent and the second reagent were prepared according to the method described in Japanese Patent Publication No. 2017-181377. 1-2. Sample pretreatment solution PBS buffer (pH 7.4) was used. 1-3. Samples Samples 4-6 (serum) described in Reference Example 2 were treated by adding PBS buffer, a sample pretreatment solution, in a 5:1 volume ratio, and reacting them at 37°C for 1 hour before measurement. 1-4. Measurement Procedure The sIL-2R concentration in each sample was measured using a Hitachi 7180 automated analyzer after mixing the sample, reagent 1, and reagent 2. Specifically, 120 μL of reagent 1 was added to 5.6 μL of the sample and incubated at 37°C for 5 minutes. Then, 40 μL of reagent 2 was added and stirred. The change in absorbance associated with aggregation formation was measured over the following 5 minutes at a primary wavelength of 570 nm and a secondary wavelength of 800 nm. The measured values ​​were calculated by applying the amount of absorbance change to a calibration curve obtained by measuring standard substances of known concentration.

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

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

[0113] [Table 3]

[0114] Based on the results from Reference Example 2, Example 2, and Comparative Example 6, we discussed the effect of IgM-specific degrading enzymes on suppressing non-specific reactions. (1) The measurement results of the control sample (sample 4) were verified. In the control sample, the measured values ​​for Reference Example 2, Example 2, and Comparative Example 6 were generally equivalent. These results demonstrate that the addition of IgMBRAZOR does not affect the measured values ​​of samples that do not exhibit a nonspecific reaction. (2) The measurement results of the discrepant samples (samples 5 and 6) were verified. The measured value for sample 5 was 306 U / mL for Reference Example 2. Comparative Example 6 was 556 U / mL, which deviated from the measured value for Reference Example 2. On the other hand, the measured value for Example 2 was 408 U / mL, showing a tendency to approach that of Reference Example 2. A similar trend was observed for sample 6. The measured value for sample 6 was 591 U / mL for Reference Example 2. Comparative Example 6 was 905 U / mL, which deviated from the measured value for Reference Example 2. On the other hand, the measurement result for Example 2 was 830 U / mL, showing a tendency to approach that of Reference Example 2. Based on the above, it was possible to suppress nonspecific reactions originating from the sample by using an IgM-specific degrading enzyme in the immunoassay method. The discrepant samples used in this study were those that could not be adequately suppressed with existing nonspecific reaction inhibitors such as anti-IgM antibodies, and it was only with the present invention's method of suppressing nonspecific reactions using an IgM-specific degrading enzyme that nonspecific reactions could be suppressed for the first time.

Claims

[Claim 1] A reagent used in an immunoassay method for measuring a target substance in a sample, An immunoassay reagent containing an enzyme that specifically degrades IgM.

Images