Immunological assays

Abstract

The present invention addresses the problem of suppressing non-specific reactions.　As a result of investigating an effect for suppressing non-specific reactions of a variety of substances using samples in which measured values greatly deviate from true values and which present non-specific reactions in order to solve the aforementioned problem, the inventors discovered that carrying out an immunoreaction in the presence of Jacalin-related lectins makes it possible to suppress non-specific reactions. Specifically, the present invention provides an immunological measurement method for immunologically measuring a to-be-measured substance in a sample, wherein the method is characterized in that an immunoreaction is carried out in the presence of Jacalin-related lectins. The present invention also provides an immunological measurement reagent that contains Jacalin-related lectins.

Description

[Technical field]

[0001] The present invention relates to an immunoassay method, and more particularly to an immunoassay method in which an immune reaction is carried out in the presence of Jacalin-related lectin. [Background technology]

[0002] One of the measurement methods in the field of diagnostic drugs is the immunoassay, which uses an immune reaction to measure a target substance present in a biological sample. Since the immunoassay uses an antigen-antibody reaction, it is a measurement method with very high specificity. Immunoassays are further broadly divided into homogeneous and heterogeneous methods. The homogeneous method is a method that specifically detects the reaction caused by the substance to be measured in a mixed solution (reaction solution) of the sample and reagent solution without B / F (binding / non-binding) separation, in contrast to the heterogeneous method, in which the main reaction is allowed to proceed and detected after completely washing and removing excess components that were not involved in the measurement reaction by the B / F separation operation. The heterogeneous method is less susceptible to the influence of nonspecific reaction substances because it involves a washing process, but it has the problem that it is complicated with many steps and takes a long time to measure. In contrast, the homogeneous method is simple because it does not involve a washing process, and the time required for measurement is short, so it is a method that is widely required in the field of clinical diagnosis, but it also has the problem of being easily influenced by nonspecific reactions. Among homogeneous methods, the measurement method using immunoagglutination (TIA) is a method for qualitatively or quantitatively detecting an analyte in a sample based on the degree of agglutination of immune complexes formed by the cross-linking action of an analyte (substance to be measured) with a specific binding partner such as an antibody. Among them, the latex immunoagglutination assay (hereinafter sometimes referred to as LTIA), which uses latex particles as an insoluble carrier to amplify the agglutination signal, is suitable for optical detection and can be easily automated, making it a highly versatile measurement method that is applied to various test items.

[0003] Meanwhile, various substances exist in biological samples, and often contain so-called non-specific factors, which are substances that cause non-specific reactions. A non-specific reaction is a phenomenon in which binding not based on a specific reaction is promoted or a specific immune reaction is hindered, which causes measurement errors. The presence of heterophilic antibodies and rheumatoid factors (RF) has been revealed as causative factors of this non-specific reaction (non-specific factors). Heterophilic antibodies are a general term for human antibodies that react with animal-derived antibodies, which are the main components of immunological measurement methods, and HAMA (human anti-mouse immunoglobulin antibody) is known as a representative example. Rheumatoid factors appear in patients with rheumatoid arthritis and share the property of reacting with animal-derived antibodies with HAMA, and both are known to be human immunoglobulin G or immunoglobulin M (Non-Patent Document 1). For example, Patent Documents 1 and 2 are known as techniques for suppressing non-specific reactions in immunoassays. Patent Document 1 discloses an immunological assay method in which an anti-human IgM antibody or an anti-human IgG antibody is added to an immunoassay system to suppress non-specific reactions caused by natural antibodies of IgM type or IgG type present in a sample. Patent Document 2 discloses a method for suppressing non-specific reactions caused by human rheumatoid factor (RF) by pretreating a sample with a sufficient amount of an animal-derived antibody capable of binding to the reactive site of RF. Examples of the animal-derived antibody include anti-human IgG antibody, anti-human IgA antibody, and / or anti-human IgM antibody. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 11-287801 [Patent Document 2] Japanese Patent Application Publication No. 07-012818 [Patent Document 3] Special Publication No. 2-503714 [Patent Document 4] Japanese Patent Application Publication No. 4-326062 [Non-patent literature]

[0005] [Non-Patent Document 1] Clinical Chemistry; Vol. 23, Supplement 175a-1~175a-10 (1994) Summary of the Invention [Problem to be solved by the invention]

[0006] Patent documents 1 and 2 are methods for suppressing non-specific reactions caused by natural antibodies and RF using anti-human IgM antibodies, anti-human IgG antibodies, and anti-human IgA antibodies. Currently, these methods are in practical use with a variety of reagents, but the use of antibodies makes the reagents expensive. Furthermore, the inventors' tests have revealed that there are also non-specific reactions that cannot be suppressed even with these antibodies. [Means for solving the problem]

[0007] An objective of the present invention is to suppress non-specific reactions in immune responses. In order to solve the above problems, the present inventors have investigated the non-specific reaction inhibitory effects of various substances, and have found that non-specific reactions can be inhibited by performing immune reactions in the presence of jacalin, which has led to the completion of the present invention. That is, the present invention has the following configuration. Jacalin is known to be used for purifying glycoproteins and capturing substances to be measured in immunological assays (Patent Documents 3 and 4), but it is not known to have a non-specific reaction inhibitory effect in immunological assays. <1> An immunological assay method for immunologically measuring a substance to be measured in a sample, the method comprising carrying out an immune reaction in the presence of jacalin-related lectin. <2> The immunoassay is based on a homogeneous method. <1> The immunoassay method according to claim 1. <3> A method based on homogeneous method is latex immunoagglutination assay. <1> or <2> The immunoassay method according to claim 1. <4> The jacalin-related lectin is jacalin; <1> ~ <3> 13. The immunoassay method according to any one of the preceding claims. <5> A method for inhibiting a non-specific reaction in an immunological measurement method for a substance to be measured in a sample, the method comprising carrying out an immune reaction in the presence of jacalin-related lectin. <6> The immunoassay is based on a homogeneous method. <5> 2. The method for suppressing a non-specific reaction according to claim 1. <7> A method based on homogeneous method is latex immunoagglutination assay. <5> or <6> 2. The method for suppressing a non-specific reaction according to claim 1. <8> The jacalin-related lectin is jacalin; <5> ~ <7> 2. The method for suppressing a non-specific reaction according to any one of the preceding claims. <9> A reagent for immunological assay comprising jacalin-related lectin. <10> The immunoassay is based on a homogeneous method. <9> The immunoassay reagent according to claim 1. <11> A method based on homogeneous method is latex immunoagglutination assay. <10> The immunoassay reagent according to claim 1. <12> The jacalin-related lectin is jacalin; <9> ~ <11> 13. The immunoassay reagent according to claim 12, <13> A reagent kit for immunological assay containing jacalin-related lectin. <14> The immunoassay is a latex immunoagglutination assay, comprising: <13> A reagent kit for immunological measurement according to claim 1. (1) A first reagent containing a jacalin-related lectin (2) a second reagent containing latex particles carrying a specific binding partner for the substance to be measured; <15> The jacalin-related lectin is jacalin; <13> or <14> A reagent kit for immunological measurement according to claim 1. <16> A non-specific reaction suppressant in an immunological assay method, comprising jacalin-related lectin as an active ingredient. <17> The jacalin-related lectin is jacalin; <16> 2. The non-specific reaction inhibitor according to claim 1. Effect of the Invention

[0008] According to the present invention, in an immunological assay, by performing an immune reaction in the presence of Jacalin-related lectin, it is possible to suppress non-specific reactions contained in a sample, thereby enabling accurate measurement. In particular, it is possible to suppress non-specific reactions that could not be sufficiently suppressed by a mixture of anti-human IgA polyclonal antibody, anti-human IgG polyclonal antibody, and anti-human IgM polyclonal antibody (hereinafter sometimes referred to as anti-human IgA+IgG+IgM polyclonal antibody), anti-human IgM polyclonal antibody, or anti-human IgA polyclonal antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] (immunological assay) The immunological assay method of the present invention is a method for immunologically measuring a substance to be measured in a sample, characterized in that an immune reaction is carried out in the presence of Jacalin-related lectin. Immunoassays are further broadly classified into homogeneous and heterogeneous methods. Homogeneous methods are methods that specifically detect the reaction caused by the substance to be measured in a mixed solution (reaction solution) of a sample and a reagent solution without B / F (binding / non-binding) separation. Specific examples include latex immunoagglutination assay (LTIA) using latex as an insoluble carrier, and immunochromatography (lateral flow type, flow-through type). Heterogeneous methods are methods that detect the main reaction by allowing it to proceed after completely washing and removing excess components that were not involved in the measurement reaction by a B / F separation operation. Specific examples include ELISA using well-shaped plates and chemiluminescence detection methods. Among them, immunoassays that do not include a step for B / F separation (homogeneous immunoassays) are more preferable because they are simple because they do not require a washing step and require a short measurement time.

[0010] (Jacalin-related lectin) Many plants contain carbohydrate-binding proteins known as lectins. These plant lectins are classified into seven protein families (Reference: Trends in Glycoscience and Glycotechnology, Vol.12 No.64 (March2000) pp.83-101). (1) Amaranthin lectin family (2) Chitin-binding lectin consisting of hevein domain (3) Phloem lectin from Cucurbitaceae (4) Jacalin-related lectins (5) Legume lectins (6) Monocotyledonous plant mannose-binding lectin (7) Type 2 RIP Jacalin-related lectins include jacalin, a galactose-specific lectin obtained from the seeds of jackfruit (Artocarpus integrifolia), and all plant lectins structurally and evolutionarily closely related. The jacalin family is divided into a galactose-specific subfamily and a mannose-specific subfamily. The jacalin-related lectin of the present invention is used to include both of these, and among these, jacalin is particularly preferred. In the present invention, jacalin will be mainly described below as a representative example of jacalin-related lectins, but other jacalin-related lectins will be described by replacing them with jacalin unless otherwise specified.

[0011] (Jacalyn) Jacalin is an aD-galactose-binding lectin extracted from the seeds of jackfruit (Artocarpus integrifolia), and is a glycoprotein of approximately 66 kDa composed of four identical subunits. In the present invention, the degree of purification of Jacalin is preferably high, but it is acceptable as long as it is present in the immune reaction system and can exert the desired non-specific inhibitory effect. In addition, Jacalin denatured by a surfactant, acid, alkali, heat treatment, etc., alone or in combination, is also included, to the extent that the desired non-specific inhibitory effect can be exerted. In addition, Jacalin that has been polymerized, Jacalin modified with a polymer compound such as PEG or a protein, Jacalin fragments decomposed by enzymes, and any of these bound to an insoluble carrier such as latex particles are also included, to the extent that the desired non-specific inhibitory effect can be exerted. In addition, Jacalin is not limited to extracts from natural products, but also includes recombinant proteins and those with appropriately modified sequences. It is known that Jacalin is used to purify IgA, but there is no example of its use for inhibiting non-specific reactions in immune reactions. Methods for making Jacalin present in an immune reaction system include adding it to an immunoassay reagent, a sample diluent, a preservation solution, or the like.

[0012] In the present invention, the concentration of Jacalin may be any concentration that does not strongly affect immune reactions and can exert the desired non-specific reaction suppression effect, and can be appropriately set by a person skilled in the art according to the type of substance to be measured and the type of specimen. The concentration of Jacalin in the immune reaction system varies depending on the composition of the reagent in the immune reaction system, but for example, the weight of Jacalin per 10 μL of specimen is 0.1 to 500 μg, preferably 1.0 to 300 μg, more preferably 2.0 to 200 μg, and even more preferably 5.0 to 100 μg, but is not limited to this concentration. In the LTIA measurement reagent, the concentration of Jacalin contained in the general first reagent is 1 to 2000 μg / mL, preferably 10 to 1500 μg / mL, more preferably 20 to 1000 μg / mL, and even more preferably 50 to 500 μg / mL, but is not limited to this concentration. Furthermore, when the sample is treated with a pretreatment solution before being subjected to LTIA measurement, Jacalin may be contained in the pretreatment solution, and the Jacalin concentration in the pretreatment solution is 0.01 to 50% by weight, preferably 0.1 to 20%, more preferably 0.1 to 10%, and even more preferably 0.2 to 5%, but is not limited to these concentrations. For example, in the case where the immunoassay reagent is a liquid reagent, the inside of the immune reaction system refers to the liquid phase in which the sample and the liquid immunoassay reagent are mixed and the immune reaction takes place. The specimen may be mixed with a liquid immunoassay reagent containing Jacalin, or the specimen may be mixed in advance with a pretreatment liquid containing Jacalin and then mixed with the immunoassay reagent. In addition, in the case where the immune reaction takes place in a solid phase such as immunochromatography, the inside of the immune reaction system refers to the solid phase in which the immune reaction between the liquid sample and the binding partner takes place. The specimen may be mixed in advance with a pretreatment liquid containing Jacalin and then dropped onto the immunochromatography test piece, or Jacalin may be dried and solidified on the sample pad, and when the specimen is dropped, Jacalin is dissolved and becomes capable of reacting with the specimen. Jacalin may be used alone, or multiple types such as modified Jacalin may be used in combination, or Jacalin bound to an insoluble carrier such as latex particles may be used. When multiple types of Jacalin are used in combination, the combined concentration of both may be within the above concentration range. When jacalin is contained in the measurement reagent of the present invention in advance, it is sufficient to contain it in the measurement reagent in advance so that the concentration in the reaction system becomes the above-mentioned concentration.

[0013] (Immunoassay Reagents) The immune reaction of the immunoassay of the present invention is carried out using an immunoassay reagent, which contains, in addition to the main reaction component, a jacalin-related lectin, which will be described later. The main reaction components include a binding partner specific to the substance to be measured, and also include insoluble carriers such as particles for immunoassay. The immunoassay reagent may contain, as a component for buffering and adjusting the pH, ionic strength, osmotic pressure, etc. of the sample, for example, acetic acid, citric acid, phosphoric acid, Tris, glycine, boric acid, carbonate, Good's buffer, or their sodium salts, potassium salts, calcium salts, etc. Furthermore, as a component for enhancing the formation of agglutination, polymers such as polyvinylpyrrolidone and phospholipid polymers may be contained. Furthermore, as a component for controlling the formation of agglutination, one or a combination of multiple commonly used components such as proteins, amino acids, sugars, metal salts, surfactants, reducing substances, and chaotropic substances may be contained.

[0014] (Particles for immunoassay) The particles for immunoassay used in the present invention may be any known particle as long as it can carry a specific binding partner for the desired target component. Latex particles using a polymeric material such as polystyrene are preferably used, but inorganic particles such as metal colloids, silica, and carbon may also be used as the particles for immunoassay in the present invention depending on the method for carrying a specific binding partner for the substance to be measured. The size of the particles for immunoassay 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.). In optical measurements using an automatic analyzer, an average particle size of 0.1 to 0.4 μm is generally used, but is not limited thereto. The average particle size of the immunoassay particles can be confirmed using a particle size distribution meter, a transmission electron microscope image, etc. The concentration of the immunoassay particles in the test solution can be appropriately selected, for example, from the range of 0.0001 mg / mL to 10 mg / mL depending on the particle size of the immunoassay particles used and the design of the entire measurement system.

[0015] When latex particles are used as the particles for immunoassay of the present invention, the synthetic polymer constituting the latex particles is not particularly limited, and examples thereof include polystyrene, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, itaconic acid polymer, and styrene-hydrophilic carboxy monomer copolymer, such as styrene-methacrylic acid copolymer, styrene-acrylic acid copolymer, and styrene-itaconic acid copolymer. Among these, preferred are styrene-methacrylic acid copolymer, styrene-itaconic acid copolymer, and styrene and styrene-styrene sulfonate copolymer. Particularly preferred are styrene and styrene-(meth)acrylic acid copolymer.

[0016] The salt of styrene sulfonate is not particularly limited, and examples thereof include sodium salt, potassium salt, lithium salt, ammonium salt, etc. These may be used alone or in combination of two or more. Among them, sodium styrene sulfonate is preferably used. The hydrophilic carboxyl monomer used in the present invention may be methacrylic acid, acrylic acid, itaconic acid, maleic acid, fumaric acid, etc. Preferably, methacrylic acid or acrylic acid can be used. The immunoagglutination method that uses latex particles as the particles for immunoassay is specifically called the latex immunoagglutination method (LTIA).

[0017] (specific binding partner) In the present invention, specific binding partners for the substance to be measured that are supported on the latex particles include proteins, peptides, amino acids, lipids, carbohydrates, nucleic acids, haptens, etc., and are not particularly limited in terms of molecular weight or origin (natural or synthetic), but include antibodies or antigens that can be used in immunological assays that utilize immune reactions.

[0018] (antibody) The antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody, and is more preferably a monoclonal antibody. As the antibody of the present invention, in addition to the whole antibody molecule, it is also possible to use a functional fragment of an antibody having antigen-antibody reaction activity. In addition to those obtained through an immunization process of common animals (mouse, goat, sheep, etc.), the antibody may be an antibody (chimeric antibody, humanized antibody, fully humanized antibody, etc.) whose amino acid sequence has been changed by genetic recombination technology to that of an animal species different from that of the animal immunized with the immunogen (substance to be measured). The functional fragment of an antibody may be F(ab'), which is a fragment having antigen-antibody reaction activity. 2 Examples of such fragments include single chain antibodies (scFv), Fab', and single chain antibodies (scFv). These functional fragments of antibodies can be produced by treating the antibodies obtained as described above with a protease (e.g., pepsin or papain).

[0019] (sample) Examples of specimens containing the substance to be measured in the present invention include human or animal blood, serum, plasma, culture supernatant, urine, cerebrospinal fluid, saliva, sweat, ascites, and cell or tissue extracts. The sample in the present invention includes not only the specimen itself obtained from a living body, but also specimens that have been pretreated, such as diluted or purified.

[0020] (Substance to be measured) The immunoassay reagent of the present invention can measure various substances contained in the sample. Examples of the measurement target substance include proteins, peptides, amino acids, lipids, carbohydrates, nucleic acids, and haptens, but there is no particular limitation as long as it is a theoretically measurable substance. Examples include CRP (C-reactive protein), Lp(a), MMP3 (matrix metalloproteinase 3), antiphospholipid antibodies, type IV collagen, PSA, BNP (brain natriuretic peptide), insulin, albumin, cystatin C, RF (rheumatoid factor), KL-6, procalcitonin, FDP, D-dimer, SF (soluble fibrin), TAT (thrombin-antithrombin III complex), PAI-1, phenytoin, phenobarbital, carbamazepine, valproic acid, and theophylline.

[0021] (Reagent composition) The immunoassay reagent of the present invention is composed of one test solution or two or more test solutions. Examples of the test solutions include a test solution made of a buffer solution for the purpose of adjusting the concentration of the substance to be measured to a suitable concentration for measurement or adjusting the environment for the antigen-antibody reaction, and a test solution containing antibody-bound particles. The Jacalin of the present invention can exert a non-specific inhibitory effect derived from the sample in the mixed liquid state during measurement, and may be contained in any or all of the constituent reagents to the extent that it does not affect the stability of the constituent reagents. In the case of a reagent for immunoassay using latex particles, an example is a reagent that contains a first reagent containing a buffer solution and a second reagent containing latex, with Jacalin-related lectin contained in either or both of them. Jacalin may also be contained in the sample pretreatment solution. The concentration of Jacalin-related lectin in each constituent reagent may be in a form that can be adjusted to the concentration in the immune reaction system when the reagent and sample are mixed at the time of measurement, and varies depending on the type of reagent.

[0022] (Latex immunoagglutination assay (LTIA) Methods for measuring target substances using LTIA can be broadly divided into two categories. The first method involves reacting the target substance with latex particles to which a specific binding partner for the target substance has been immobilized to form a sandwich-type immune complex, and measuring the target substance based on the degree of agglutination of the latex particles associated with immune complex formation. The second method involves adding proteins or the like to which multiple substances to be measured or their analogs (including fragments of these) are immobilized to a reagent, and allowing these to compete with the substances to be measured in the sample to inhibit the formation of an immune complex 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 the substance to be measured (antigen) is measured from the degree of inhibition of agglutination of the latex particles that accompanies the inhibition of immune complex formation. The substance to be measured and the specific binding partner for the substance to be measured can be selected from any substances depending on the purpose. For example, if the substance to be measured is an antigen, an antibody such as a polyclonal antibody or a monoclonal antibody (including recombinant antibodies and functional fragments of each antibody) can be selected as the specific binding partner for the substance to be measured. If the substance to be measured is an antibody, an antigen such as a natural or recombinant antigen can be selected as the specific binding partner for the substance to be measured. The present invention can be used in any of the above methods, and specifically, the following steps are exemplified. (1) contacting a sample with jacalin in a liquid phase; (2) After the step (1), a step of adding the latex particles to a liquid phase. (3) After the step (2), a step of measuring the agglutination reaction between the target substance and the latex particles. 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 or separation step."

[0023] The measurement method of LTIA is to measure the test substance by optically or electrochemically observing the degree of agglutination. Examples of optical observation methods include methods (endpoint method, rate method, etc.) that measure scattered light intensity, absorbance, or transmitted light intensity with optical equipment. The measured value of absorbance, etc. obtained by measuring a sample is compared with the measured value of absorbance, etc. obtained by measuring a standard substance (a sample with a known concentration of the substance to be measured), to calculate the concentration (quantitative value) of the substance to be measured contained in the sample. The measurement of absorbance, etc. of transmitted light or scattered light may be a one-wavelength measurement or a two-wavelength measurement (the difference or ratio of two wavelengths). The measurement wavelength is generally selected from 500 nm to 900 nm.

[0024] The measurement of the substance to be measured in the sample of the present invention 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 (dedicated machine). In addition, the measurement is preferably performed by a method that involves multiple operational steps, such as a two-step method (two-reagent method).

[0025] (Latex particles carrying specific binding partners) A specific binding partner for the substance to be measured can be immobilized and supported on latex particles by known methods such as physical adsorption, chemical binding, or a combination of these. In the case of the physical adsorption method, the measurement can be carried out according to a known method, for example by mixing and contacting a specific binding partner for the substance to be measured with latex particles in a solution such as a buffer solution, or by contacting a specific binding partner for the substance to be measured dissolved in a buffer solution or the like with a carrier. In addition, when using a chemical binding method, the method can be carried out according to known methods described in, for example, "Clinical Pathology Special Issue No. 53: Immunoassays for Clinical Tests - Techniques and Applications" edited by the Japanese Society of Clinical Pathology, Clinical Pathology Publishing Society, published in 1983; "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 reacting the amino groups, carboxyl groups, thiol groups, aldehyde groups, hydroxyl groups, etc. of the specific binding partner for the substance to be measured and the carrier with the bivalent cross-linking reagent.

[0026] It is preferable that the specific binding partners for the specific substance carried by the latex particles are of multiple types in order to form a sandwich. When the specific substance has multiple antibody recognition sites, the specific binding partner may be of one type. For example, when the specific binding partner is a monoclonal antibody, multiple monoclonal antibodies with different recognition sites are used. Also, when the specific binding partner is a polyclonal antibody, the specific binding partner may be a polyclonal antibody derived from one type of antiserum, or may be derived from multiple types of antisera. Also, a monoclonal antibody and a polyclonal antibody may be used in combination.

[0027] If it is necessary to perform a treatment to suppress spontaneous aggregation or nonspecific reactions of the latex particles, the surface of the latex particles may be treated by a known method, such as by contacting and coating with a protein, such as bovine serum albumin (BSA), casein, gelatin, egg white albumin or a salt thereof, a surfactant, or skim milk powder, to perform a blocking treatment (masking treatment) of the carrier.

[0028] (Method of suppressing non-specific reactions) It often happens that some component contained in a biological sample causes agglutination that should not occur (positive measurement error) or agglutination that should occur does not occur (negative measurement error) in immunoassay particles that have a specific binding partner for the substance to be measured immobilized thereon. These are called non-specific reactions and are known to cause various measurement errors. In the present invention, suppressing non-specific reactions means acting on factors (non-specific factors) that cause the above-mentioned non-specific reactions in a biological sample and suppressing the influence of reactions other than immune reactions on the measurement. The present invention can suppress non-specific reactions by performing immune reactions in the presence of Jacalin-related lectins.

[0029] (Reagent Kit) The reagent kit of the present invention is characterized in that it contains at least Jacalin-related lectin in its kit configuration. The kit configuration includes not only immunoassay reagents, but also sample dilution solutions, sample extracts, etc., and the Jacalin-related lectin of the present invention can be contained in one or more of these. In particular, when the immunological assay is a latex immunoagglutination assay, it is preferable that the reagent composition includes the following: (1) A first reagent containing a jacalin-related lectin (2) a second reagent containing latex particles carrying a specific binding partner for the substance to be measured; The first reagent typically contains a buffer solution, and the concentration of the Jacalin-related lectin in the buffer solution may be adjusted to the preferred Jacalin-related lectin concentration in the mixed state of the reagent and the sample at the time of measurement, and may vary depending on the type of the reagent. The Jacalin-related lectin may be contained in the second reagent in addition to the first reagent. In addition to the above, the kit also includes instructions for use and sample collection tools (collection pipettes, syringes, cotton swabs, filtration filters, etc.).

[0030] (Non-specific reaction inhibitors) The non-specific reaction inhibitor of the present invention is a drug for suppressing non-specific reactions derived from a sample in an immunological measurement method, and contains at least Jacalin-related lectin as an active ingredient. The composition containing Jacalin in the above-mentioned reagent kit can be used as it is.

[0031] (Other Reagent Components) The immunoassay reagent of the present invention may contain a polymer such as polyethylene glycol, polyvinylpyrrolidone, or a phospholipid polymer as a component that enhances the agglutination of insoluble carrier particles. In addition, as a component that controls the formation of aggregates, it may contain one or a combination of multiple commonly used components such as proteins, amino acids, sugars, metal salts, surfactants, reducing substances, and chaotropic substances. The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples. EXAMPLES

[0032] The following describes a test in which soluble interleukin 2 receptor (hereinafter referred to as sIL-2R) was measured using a reagent containing Jacalin and other substances that inhibit non-specific reactions according to the present invention. The samples used were those that exhibited non-specific reactions and whose measured values ​​deviated significantly from the true values. Specifically, the test samples used were sample A, which was believed to be the main cause of non-specific reactions to be IgA, sample B, which was believed to be IgG, and sample C, which was believed to be IgM.

[0033] [Reference Example 1] (Obtaining anti-sIL-2R monoclonal antibody) A monoclonal antibody against sIL-2R was obtained by the method described in WO2017 / 057622. Furthermore, a combination of antibodies applicable to sandwich immunoassay (clone numbers 92212 and 92204R) was selected according to the method described in WO2017 / 057622.

[0034] [Comparative Example 1] (Measurement by ELISA) The anti-sIL-2R monoclonal antibody (clone number 92212) obtained in Reference Example 1 was dissolved in 20 mM phosphate buffer (pH 7.2; hereinafter referred to as PBS) containing 150 mM sodium chloride to a concentration of 10 μg / mL, and 100 μL of the solution was dispensed into each well of a 96-well microplate and allowed to stand overnight at 4° C. Each well was washed three times with 400 μL of PBS containing 0.05% Tween (registered trademark) 20 (hereinafter referred to as PBST), and then 200 μL of PBST containing 1% bovine serum albumin (hereinafter referred to as BSA-PBST) was added and blocked at room temperature for 1 hour. This was used as an ELISA plate. Each well of the ELISA plate was washed three times with 400 μL of PBST, and then 100 μL of a sample diluted 40-fold with BSA-PBST was added to each well and left to stand at room temperature for 1 hour. Each well was washed three times with 400 μL of PBST, and then 100 μL of a biotin-labeled anti-sIL-2R monoclonal antibody (clone number 92204R) diluted to 0.50 μg / mL with BSA-PBST was dispensed into each well and left to stand at room temperature for 1 hour. Each well was washed three times with 400 μL of PBST, and then 100 μL of HRP-labeled streptavidin (ThermoFishier Scientific) diluted to 0.20 μg / mL with BSA-PBST was dispensed into each well and left to stand at room temperature for 30 minutes. Each well was washed three times with 400 μL of PBST, after which 50 μL of citrate buffer (pH 5.0) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added and allowed to stand at room temperature for 10 minutes. The enzyme reaction was then stopped by adding 50 μL of 4.5 N sulfuric acid, and the absorbance at a wavelength of 492 nm was measured. A standard substance with a known concentration was used as a calibrator to calculate the sIL-2R value in the test sample. The measurement results are shown in the table. Since the ELISA method involves B / F separation, the sIL-2R measurement value obtained in this Comparative Example 1 was considered to be the true value of the test sample.

[0035] [Comparative Example 2] (Measurement by LTIA: No addition of non-specific reaction inhibitors) 1. Reagents The first and second reagents were prepared according to the method described in JP 2017-181377 A.

[0036] 2.Measurement method The first and second reagents were combined and used with a Hitachi 7180 automatic analyzer to measure the sIL-2R concentration in the sample examined in Comparative Example 1. Specifically, 120 μL of the first reagent was added to 5.6 μL of the sample and incubated at 37° C. for 5 minutes, after which 40 μL of the second reagent was added and stirred. The change in absorbance associated with the formation of aggregates was measured over the next 5 minutes at a main wavelength of 570 nm and a sub-wavelength of 800 nm, and the change in absorbance was applied to a calibration curve obtained by measuring a standard substance of known concentration to calculate the sIL-2R value. The measurement results are shown in the table.

[0037] [Comparative Example 3] (Measurement by LTIA: Addition of anti-human IgA + IgG + IgM polyclonal antibodies) The measurement was performed in the same manner as in Comparative Example 2, except that a mixture of anti-human IgA polyclonal antibody, anti-human IgG polyclonal antibody, and anti-human IgM polyclonal antibody (Sera Care, KPL Affinity Purified Antibody to Human IgA+IgG+IgM(H+L)[Goat] MSA) was added to the first reagent shown in Comparative Example 2 to a final concentration of 100 μg / mL. The measurement results are shown in the table.

[0038] [Comparative Example 4] (Measurement by LTIA: Addition of anti-human IgM polyclonal antibody) The measurement was performed in the same manner as in Comparative Example 2, except that an anti-human IgM polyclonal antibody (Goat Anti-Human IgM, WuXi AppTec) was added to the first reagent to a final concentration of 100 μg / mL. The measurement results are shown in Table 1.

[0039] [Comparative Example 5] (Measurement by LTIA: Addition of anti-human IgA polyclonal antibody) The measurement was performed in the same manner as in Comparative Example 2, except that anti-human IgA polyclonal antibody (Goat Anti-Human IgA, WuXi AppTec) was added to the first reagent to a final concentration of 100 μg / mL. The measurement results are shown in Table 1.

[0040] [Example 1] (Measurement by LTIA: Jacalin added) The measurements were performed in the same manner as in Comparative Example 2, except that Jacalin-I (manufactured by Extracellular Matrix Research Institute, Ltd.) was added to the first reagent shown in Comparative Example 2 to a final concentration of 100 or 400 μg / mL. The measurement results are shown in Table 1.

[0041] [Table 1]

[0042] (Results and Discussion) For sample A, the sIL-2R value by ELISA was 266 U / mL (Comparative Example 1). In the LTIA, when no non-specific reaction inhibitor was added, the measured value for this sample was 1179 U / mL (Comparative Example 2), which was significantly different from the value for Comparative Example 1. This suggested that the non-specific reaction was mainly caused by the non-specific factor IgA contained in the sample. In contrast, the measured value when a mixture of anti-human IgA polyclonal antibody, anti-human IgG polyclonal antibody, and anti-human IgM polyclonal antibody was added was 1047 U / mL (Comparative Example 3), and the measured value when anti-human IgM polyclonal antibody was added was 1160 U / mL (Comparative Example 4), which were values ​​equivalent to those in Comparative Example 2, and no inhibitory effect on non-specific reactions was observed. The measured value when anti-human IgA polyclonal antibody was added was 564 U / mL (Comparative Example 5), and although an inhibitory effect on non-specific reactions was observed, this was still about twice as high as that in Comparative Example 1, indicating that the inhibitory effect of this substance on non-specific reactions was insufficient. On the other hand, when 100 μg / mL of Jacalin of the present invention was added, the measured value was 342 U / mL, and when 400 μg / mL of Jacalin was added, the measured value was 337 U / mL, which were close to the values ​​in Comparative Example 1. Thus, it was demonstrated that Jacalin of the present invention has the effect of suppressing non-specific reactions caused by the non-specific factor IgA in the sample. Similarly, in samples B and C, the measured values ​​when Jacalin of the present invention was added were closer to the sIL-2R value measured by ELISA (Comparative Example 1) than when no non-specific reaction inhibitor was added (Comparative Example 2) or when other non-specific reaction inhibitors were added (Comparative Examples 3, 4, and 5). This demonstrates that Jacalin of the present invention has the effect of suppressing non-specific reactions mainly caused by non-specific factors IgG and IgM in the sample. It is known that Jacalin is used to purify glycoproteins and capture substances to be measured in immunological assay methods (Patent Documents 3 and 4), but it was not known that it exhibited an effect of suppressing non-specific reactions in immunological assay methods, and this finding was discovered for the first time by the present invention. In particular, it is of great significance that Jacalin of the present invention was able to suppress non-specific reactions that could not be sufficiently suppressed even by anti-human IgA+IgG+IgM polyclonal antibodies, anti-human IgM polyclonal antibodies, or anti-human IgA polyclonal antibodies. [Industrial Applicability]

[0043] According to the present invention, by carrying out an immune reaction in the presence of jacalin, it is possible to realize an accurate immunoassay method even when a sample contains non-specific factors.

Claims

1. A method for immunologically measuring a substance to be measured in a sample, the method being characterized in that the immune reaction is carried out in the presence of jacalin-related lectin as a non-specific reaction inhibitor.

2. 2. The immunoassay method according to claim 1, which is based on a homogeneous method.

3. 3. The immunological assay method according to claim 1 or 2, wherein the homogeneous method is a latex immunoagglutination assay.

4. The immunological assay method according to any one of claims 1 to 3, wherein the jacalin-related lectin is jacalin.

5. 1. A method for inhibiting a non-specific reaction in an immunological measurement method for a substance to be measured in a sample, comprising: A method for suppressing non-specific reactions, comprising carrying out an immune reaction in the presence of jacalin-related lectin as a non-specific reaction suppressor.

6. 6. The method for inhibiting a non-specific reaction according to claim 5, wherein the immunological assay is a method based on a homogeneous method.

7. 7. The method for inhibiting a non-specific reaction according to claim 5 or 6, wherein the method based on a homogeneous method is a latex immunoagglutination assay.

8. The method for inhibiting a non-specific reaction according to any one of claims 5 to 7, wherein the jacalin-related lectin is jacalin.

9. A reagent for immunological assays comprising jacalin-related lectin as a non-specific reaction inhibitor.

10. The reagent for immunoassay according to claim 9, wherein the immunoassay is based on a homogeneous method.

11. The reagent for immunoassay according to claim 10, wherein the homogeneous method is a latex immunoagglutination assay.

12. The reagent for immunoassay according to any one of claims 9 to 11, wherein the jacalin-related lectin is jacalin.

13. A reagent kit for immunological assays comprising jacalin-related lectin as a non-specific reaction inhibitor.

14. The reagent kit for an immunoassay according to claim 13, wherein the immunoassay is a latex immunoagglutination assay, and comprises: (1) A first reagent containing a jacalin-related lectin as a non-specific reaction inhibitor (2) A second reagent containing latex particles carrying a specific binding partner for the substance to be measured

15. The reagent kit for immunological assay according to claim 13 or 14, wherein the jacalin-related lectin is jacalin.

16. A non-specific reaction suppressant in an immunological assay method, comprising jacalin-related lectin as an active ingredient.

17. The non-specific reaction inhibitor according to claim 16, wherein the jacalin-related lectin is jacalin.

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