Immunological measurement method, non-specific reaction inhibition method, immunological measurement reagent, immunological measurement kit, composition, non-specific reaction inhibitor, and use

Abstract

The present invention relates to an immunological measurement method for immunological measurement of a substance to be measured in a sample, an immune reaction is carried out in the presence of an anti-immunoglobulin L chain lambda monoclonal antibody and an anti-immunoglobulin L chain kappa monoclonal antibody, in the presence of an anti-immunoglobulin L chain lambda monoclonal antibody or in the presence of an anti-immunoglobulin L chain kappa monoclonal antibody; this immunological measurement method can suppress non-specific reactions that cannot be eliminated by conventional non-specific reaction inhibitors.

Description

Technical Field

[0001] The present invention relates to an immunological assay method, a non-specific reaction inhibition method, an immunological assay reagent, an immunological assay kit, a composition, a non-specific reaction inhibitor and uses.

[0002] This application claims priority based on Japanese Patent Application No. 2022-136075 filed in Japan on August 29, 2022, the contents of which are incorporated herein by reference. Background Art

[0003] As a measurement method in the field of diagnostic drugs, there is an immunological measurement method that utilizes an immune reaction to measure a measurement target substance present in a biological sample. Since the immunological measurement method utilizes an antigen-antibody reaction, it is a measurement method with very high specificity.

[0004] Various substances exist in biological samples, and thus may cause measurement errors by promoting binding that is not based on specific reactions or inhibiting specific immune reactions. Such phenomena are called non-specific reactions, and substances that cause non-specific reactions are called non-specific factors.

[0005] As non-specific factors, heterophilic antibodies and rheumatoid factors (RF) are known to exist. Heterophilic antibodies are a general term for human antibodies that are reactive to animal-derived antibodies that are responsible for the main reaction of immunological measurement methods, and heterophilic antibodies represented by HAMA (human anti-mouse immunoglobulin antibodies) are known. As for rheumatoid factors, they appear in patients with rheumatoid arthritis and have the same property as HAMA in that they are reactive to animal-derived antibodies, and their entities are known to be human immunoglobulin G or immunoglobulin M (Non-Patent Document 1).

[0006] As a technique for suppressing non-specific reactions in immunological measurement methods, for example, Patent Document 1 and Patent Document 2 are known.

[0007] Patent Document 1 discloses a method of pre-treating a sample with a sufficient amount of an animal-derived antibody having a binding ability to a reactive site (Fab) of human rheumatoid factor (RF) to suppress a non-specific reaction caused by RF. Examples of such animal-derived antibodies include anti-human immunoglobulin Fab antibodies, anti-human IgG antibodies (Fab specific), anti-human IgA antibodies (Fab specific), and anti-human IgM antibodies (Fab specific).

[0008] Patent Document 2 discloses a method of preparing a polyclonal antibody against natural IgM antibodies from an animal of the same species as the antibody used for measurement, and adding the polyclonal antibody against natural IgM antibodies to suppress non-specific reactions.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent Application Laid-Open No. 07-012818

[0012] Patent Document 2: Japanese Patent Application Laid-Open No. 11-287801

[0013] Non-patent literature

[0014] Non-patent document 1: Clinical Chemistry; Supplement to Vol. 23, pp. 175a-1 to 175a-10 (1994) Summary of the invention

[0015] Technical problem solved by the invention

[0016] As shown in Patent Document 1, it is known that natural antibodies, anti-human IgM polyclonal antibodies, anti-human IgG polyclonal antibodies, and anti-human IgA polyclonal antibodies for inhibiting nonspecific reactions caused by RF are nonspecific reaction inhibitors currently in practical use in the form of various reagents, and there are also nonspecific reactions that cannot be inhibited by this method.

[0017] Furthermore, as shown in Patent Document 2, polyclonal antibodies against natural IgM antibodies must be prepared from animals homologous to the antibodies used for measurement, and there are restrictions on the preparation method and the measurement antibodies used.

[0018] An object of the present invention is to provide an immunological assay method using a novel nonspecific reaction inhibitor capable of suppressing nonspecific reactions that cannot be eliminated by conventional nonspecific reaction inhibitors.

[0019] Technical means of solving problems

[0020] In order to solve the above-mentioned problems, the present inventors have studied the non-specific reaction inhibitory effect of various substances and found that non-specific reactions can be suppressed by carrying out immune reaction in the presence of anti-immunoglobulin L chain λ (lambda) monoclonal antibody and anti-immunoglobulin L chain κ (kappa) monoclonal antibody, in the presence of anti-immunoglobulin L chain λ monoclonal antibody or in the presence of anti-immunoglobulin L chain κ monoclonal antibody, thereby completing the present invention.

[0021] Specifically, the present invention has the following configurations.

[0022] [1] An immunological assay method, wherein:

[0023] In the method of immunologically measuring a substance to be measured in a sample, an immune reaction is carried out in the presence of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody, in the presence of an anti-immunoglobulin L chain λ monoclonal antibody, or in the presence of an anti-immunoglobulin L chain κ monoclonal antibody.

[0024] However, the substance to be measured does not include anti-immunoglobulin L chain λ antibody and anti-immunoglobulin L chain κ antibody.

[0025] [2] The immunological assay method according to [1], which is an immunological assay method based on a homogeneous method or an immunological assay method based on a heterogeneous method.

[0026] [3] The immunological assay method according to [2], comprising:

[0027] a step of contacting a sample containing a substance to be measured with the anti-immunoglobulin L chain λ monoclonal antibody or the anti-immunoglobulin L chain κ monoclonal antibody;

[0028] a step of adding a specific binding partner for the substance to be measured; and

[0029] A step of detecting a signal derived from a reaction between the analyte in the solution and the specific binding partner.

[0030] [4] The immunological assay method according to [2], wherein:

[0031] The immunological determination method based on the homogeneous method is latex immunoturbidimetry.

[0032] [5] The immunological assay method according to [4], comprising:

[0033] a step of contacting a sample containing a substance to be measured with the anti-immunoglobulin L chain λ monoclonal antibody or the anti-immunoglobulin L chain κ monoclonal antibody in a solution;

[0034] a step of adding latex particles carrying a specific binding partner for the substance to be measured to the solution; and

[0035] A step of optically detecting the degree of aggregation of the latex particles in the solution.

[0036] [6] A method for suppressing a non-specific reaction in a method for immunologically measuring a substance to be measured in a sample, wherein:

[0037] The immune reaction is carried out in the presence of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody, in the presence of an anti-immunoglobulin L chain λ monoclonal antibody, or in the presence of an anti-immunoglobulin L chain κ monoclonal antibody.

[0038] [7] The method for inhibiting a nonspecific reaction according to [6], wherein:

[0039] The method for performing immunological assay is an immunological assay method based on a homogeneous method or an immunological assay method based on a heterogeneous method.

[0040] [8] The method for inhibiting a nonspecific reaction according to [7], wherein:

[0041] The immunological determination method based on the homogeneous method is latex immunoturbidimetry.

[0042] [9] The method for inhibiting a nonspecific reaction according to [8], comprising:

[0043] a step of contacting a sample containing a substance to be measured with the anti-immunoglobulin L chain λ monoclonal antibody or the anti-immunoglobulin L chain κ monoclonal antibody in a solution;

[0044] a step of adding latex particles carrying a specific binding partner for the substance to be measured to the solution; and

[0045] A step of optically detecting the degree of aggregation of the latex particles in the solution.

[0046]

[10] An immunological assay reagent comprising either or both of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody.

[0047]

[11] The immunological assay reagent according to

[10] , which is used in an immunological assay method based on a homogeneous method or an immunological assay method based on a heterogeneous method.

[0048]

[12] The immunoassay reagent according to

[11] , wherein

[0049] The immunological determination method based on the homogeneous method is latex immunoturbidimetry.

[0050]

[13] An immunological assay kit comprising either or both of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody.

[0051]

[14] The immunological assay kit according to

[13] , which is used for an immunological assay method based on a homogeneous method or an immunological assay method based on a heterogeneous method.

[0052]

[15] The immunoassay kit according to

[14] , wherein

[0053] The immunological determination method based on the homogeneous method is latex immunoturbidimetry.

[0054]

[16] The immunoassay kit according to

[14] , wherein

[0055] The homogeneous method is latex immunoturbidimetry.

[0056] The immunological assay kit comprises the following reagents:

[0057] (1) a first reagent comprising either or both of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody;

[0058] (2) A second reagent comprising latex particles supporting a specific binding partner for a substance to be measured.

[0059]

[17] A composition comprising either or both of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody.

[0060]

[18] A non-specific reaction inhibitor for an immunological assay method, comprising as an active ingredient either or both of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody.

[0061]

[19] Use of the composition described in

[17] as a non-specific reaction inhibitor in an immunological assay method.

[0062] Effects of the Invention

[0063] According to the present invention, there can be provided an immunoassay method using a novel nonspecific reaction inhibitor capable of suppressing nonspecific reactions that cannot be eliminated by conventional nonspecific reaction inhibitors.

[0064] In the immunological measurement method of the present invention, non-specific reactions that cannot be suppressed even with commercially available non-specific reaction inhibitors can be suppressed, and accurate measurement can be performed. DETAILED DESCRIPTION

[0065] <Immunological Assay Methods>

[0066] The immunological assay method of the present invention is a method for immunologically assaying an assay target substance in a sample, wherein an immune reaction is carried out in the presence of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody, in the presence of an anti-immunoglobulin L chain λ monoclonal antibody, or in the presence of an anti-immunoglobulin L chain κ monoclonal antibody. In other words, it is a method for immunologically assaying an assay target substance in a sample using a specific binding partner other than an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody, an anti-immunoglobulin L chain λ monoclonal antibody, or an anti-immunoglobulin L chain κ monoclonal antibody in the presence of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody, in the presence of an anti-immunoglobulin L chain λ monoclonal antibody, or in the presence of an anti-immunoglobulin L chain κ monoclonal antibody as a non-specific reaction inhibitor.

[0067] Immunological assay methods are further broadly divided into homogeneous and heterogeneous methods.

[0068] The homogeneous method refers to a method of specifically detecting the binding reaction of the substance to be measured without performing B / F (bound / unbound) separation in a mixed solution (reaction solution) of a sample and a reagent solution, while the heterogeneous method refers to a method of detecting the main reaction after washing / removing the remaining components that do not participate in the measurement reaction through a B / F separation operation.

[0069] The heterogeneous method has the problem that it needs more steps and takes time to measure because it has a cleaning process, but it has the advantage that it is relatively less susceptible to the influence of non-specific reaction substances. The homogeneous method, on the other hand, has the problem that it does not need a cleaning process and is easily affected by non-specific reactions, but it has fewer and simpler steps and takes less time to measure, so it is a method widely needed in the field of clinical diagnosis.

[0070] As homogeneous methods, there can be cited the measurement method using immunoagglutination method (TIA) and immunochromatography (lateral flow, flow-through). As for TIA, it is a method for qualitatively or quantitatively detecting the analyte in the sample based on the degree of agglutination of the immune complex formed by the cross-linking of the analyte (substance to be measured) with a specific binding partner such as an antibody. In particular, the latex immunoturbidimetry (hereinafter sometimes referred to as LTIA) using latex particles as an insoluble carrier to amplify the agglutination signal is suitable for optical detection and easy automation, so it is a highly versatile measurement method applicable to various inspection items.

[0071] Examples of the heterogeneous method include an ELISA method using a well plate and a chemiluminescence method.

[0072] The present invention can be applied to any of the above-mentioned immunological assay methods, but the homogeneous method which is relatively easily affected by non-specific reactions can be expected to achieve better results and is therefore preferred.

[0073] (anti-immunoglobulin L chain λ monoclonal antibody, anti-immunoglobulin L chain κ monoclonal antibody)

[0074] In the present invention, an anti-immunoglobulin L chain λ monoclonal antibody and / or an anti-immunoglobulin L chain κ monoclonal antibody is used as an active ingredient for suppressing non-specific reactions.

[0075] The anti-immunoglobulin L chain λ monoclonal antibody is an antibody that reacts with the λ chain of the immunoglobulin L chain, and may be an antibody against a free L chain (including Bence Jones protein) λ chain or an antibody against a non-free L chain λ chain.

[0076] The anti-immunoglobulin L chain κ monoclonal antibody is an antibody that reacts with the κ chain of the immunoglobulin L chain, and may be an antibody against a free L chain κ chain or an antibody against a non-free L chain κ chain.

[0077] Free immunoglobulin L chain refers to a monomer or dimer of immunoglobulin L chain that is not associated with an immunoglobulin H chain (heavy chain). Immunoglobulin L chains are divided into two types, κ chain and λ chain, due to differences in antigenicity. In this specification, anti-immunoglobulin L chain λ antibody and anti-immunoglobulin L chain κ antibody are referred to as anti-L chain λ antibody and anti-L chain κ antibody, respectively, hereinafter.

[0078] In addition, unless otherwise specified in the present specification, anti-L chain λ antibody and anti-L chain κ antibody are intended to include both monoclonal antibodies and polyclonal antibodies.

[0079] In addition, in this specification, κ may be represented as kappa, and λ may be represented as lambda.

[0080] Furthermore, as the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody used in the present invention, in addition to the whole antibody molecule, a functional fragment of the antibody having reactivity with the antigen may also be used.

[0081] As for the anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody used in the present invention, in addition to the monoclonal antibody obtained by immunizing a general animal, it can also be a monoclonal antibody obtained by a method such as phage display technology. Usually, antibody-producing cells are fused with myeloma-derived cell lines to prepare fused cells (hybridomas) with autonomous proliferation ability, and fused cells that only produce antibodies reactive to the target antigen are selected (screened), and antibody-producing hybridomas are established by singularizing (cloning). The target monoclonal antibody is obtained by purifying the cell-derived antibody or transplanting the cell-derived antibody into mice to recover ascites.

[0082] Monoclonal antibodies are antibodies having amino acid sequences derived from different animal species, specifically, chimeric antibodies, humanized antibodies, or fully human antibodies. In addition, as functional fragments of antibodies, F(ab')2, Fab', single-chain antibodies (scFv), VHH (variable domain of heavy chain of heavy chain antibody) antibodies, IgNAR (new antigen receptor) antibodies, etc., which are fragments having antigen-antibody reactivity, can be cited. The functional fragments of these antibodies can be produced by treating the antibodies obtained as described above with protease (e.g., pepsin, papain, etc.), or by utilizing gene recombination technology. In the present invention, unless otherwise specified, anti-L chain λ monoclonal antibodies and anti-L chain κ monoclonal antibodies may contain functional fragments.

[0083] As described above, the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody may be used in combination, and other known non-specific reaction inhibitors such as anti-human IgM antibodies may be combined separately or in combination.

[0084] In this specification, the "reaction" of an antibody with an antigen and the "recognition" of an antibody with an antigen are used synonymously and are not limited to these examples and need to be interpreted in the broadest sense. As for whether an antibody and an antigen (compound) "react" or not, in addition to being carried out by antigen solid phase ELISA, competitive ELISA, sandwich ELISA, etc., it can also be carried out by a method (SPR method) based on the principle of surface plasmon resonance. The SPR method can be carried out using a commercially available device, sensor, and reagent named Biacore (registered trademark).

[0085] As for the anti-L chain λ antibody and anti-L chain κ antibody used in the present invention, they can be prepared by dissolving commercially available human-derived immunoglobulins as antigens (immunogens) in solvents such as phosphate-buffered saline, and administering the solution to animals for immunization. After adding a suitable adjuvant to the solution as needed, an emulsion can be used for immunization. As adjuvants, in addition to general adjuvants such as water-in-oil emulsions, water-in-oil-in-water emulsions, water-in-oil emulsions, liposomes, aluminum hydroxide gels, proteins derived from biological components, peptide properties, etc. can also be used. For example, Freund's incomplete adjuvant or Freund's complete adjuvant can be used appropriately. The route of administration, dosage, and administration period of the adjuvant are not particularly limited, and it is best to select appropriately to enhance the desired immune response in animals immunized with antigens.

[0086] The type of animal used in the immunization is not particularly limited, preferably a mammal, for example, mice, rats, cattle, rabbits, goats, sheep, alpacas, etc. can be used, and more preferably mice or rats can be used. The immunization of the animal can be carried out according to the usual method, for example, the immunization can be carried out by injecting a solution of the antigen into the subcutaneous, intradermal, intravenous or intraperitoneal cavity of the animal, wherein the solution of the antigen is preferably a mixture with an adjuvant. The immune response generally varies according to the type and system of the immunized animal, so the immunization schedule is preferably appropriately set according to the animal used. Antigen administration is preferably repeated several times after the initial immunization.

[0087] To obtain a monoclonal antibody, the following operation is then performed, but is not limited thereto. The method for producing a monoclonal antibody itself is well known and is commonly used in the art, so a person skilled in the art can easily produce the monoclonal antibody used in the immunological analysis method of the present invention (for example, see Chapter 6 of Reference 1, etc.).

[0088] After the final immunization, the spleen cells or lymph node cells as antibody-producing cells can be extracted from the immunized animal, and the cell lines derived from myeloma with higher proliferation capacity can be used to carry out cell fusion to prepare hybridomas. Cells with higher antibody production capacity (quality / quantity) are preferably used in cell fusion, and the cell lines derived from myeloma are preferably compatible with the animal from which the antibody-producing cells to be fused are derived. Cell fusion can be carried out according to the known methods in the technical field to which the present invention belongs, for example, polyethylene glycol method, Sendai virus method, method using electric current, etc. can be adopted. The hybridoma obtained can be propagated according to the conditions common to this area, and the properties of the antibodies produced can be confirmed and the desired hybridoma can be selected. The cloning of hybridomas can be carried out by well-known methods such as limiting dilution method and soft agar method.

[0089] The selection of hybridomas can be carried out efficiently at the stage of selection by taking into account the conditions under which the produced antibodies are used in actual assays. For example, hybridomas producing the desired antibodies can be selected more efficiently by reacting antibodies obtained by immunizing animals with antigens (L chain λ chain or L chain κ chain) immobilized on a solid phase and comparing the reactivity with that in the absence of the antigen as a control.

[0090] More specifically, the case where the antibody reacts with the λ chains of two or more classes (e.g., IgA and IgG, etc.) can be judged as λ chain specific. Similarly, the case where the antibody reacts with the κ chains of two or more classes (e.g., IgA and IgG, etc.) can be judged as κ chain specific. On the other hand, when the antibody reacts with a certain class (e.g., IgA) regardless of the λ chain or the κ chain, and does not react with the different class (e.g., IgG), it is judged that the antibody has no specificity for the κ chain and the λ chain, but has specificity for the former certain class (IgA in this example).

[0091] After the cloning step, the ability of the produced antibody to bind to the L chain λ chain or L chain κ chain can be tested by using ELISA, RIA, fluorescent antibody method, etc., to confirm whether the selected hybridoma produces a monoclonal antibody with desired properties.

[0092] Monoclonal antibodies with desired characteristics can be produced by mass culturing the hybridomas selected as described above. The method of mass culturing is not particularly limited, and for example, a method of culturing hybridomas in a suitable culture medium to produce monoclonal antibodies in the culture medium; a method of injecting hybridomas into the peritoneal cavity of a mammal and causing them to proliferate to produce antibodies in ascites, etc. The purification of monoclonal antibodies can be appropriately combined with the purification methods described above for purifying antibodies from antiserum, such as DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, etc.

[0093] In the present invention, methods for making both or either of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody present in the immune reaction system include a method of using as one of the reagent components of an immunoassay reagent, and a method of adding to a diluent or pretreatment solution of a sample.

[0094] The immunoreaction system refers to a liquid phase in which a sample and a liquid immunoassay reagent are mixed to cause an immunoreaction, for example, when the immunoassay reagent is a liquid reagent.

[0095] For example, in the case of the LTIA method, the sample can be mixed with an LTIA assay reagent containing both or either an anti-L chain λ monoclonal antibody and an anti-L chain κ monoclonal antibody, or the sample can be pre-mixed with a pretreatment solution containing both or either an anti-L chain λ monoclonal antibody and an anti-L chain κ monoclonal antibody and then mixed with the immunoassay reagent.

[0096] In the case of the ELISA method, the sample may be pre-mixed with a pretreatment solution containing both or either of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody and then added dropwise to the microplate, or the sample may be mixed with a solution containing both or either of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody and a detection antibody and then added dropwise to the microplate.

[0097] In addition, in the case of a chemiluminescent reagent, the sample may be pre-mixed with a pretreatment solution containing both or either an anti-L chain λ monoclonal antibody and an anti-L chain κ monoclonal antibody and then mixed with the immunoassay reagent. The immunoassay reagent (for example, a solution containing a detection antibody or antigen, magnetic particles) may contain both or either an anti-L chain λ monoclonal antibody and an anti-L chain κ monoclonal antibody.

[0098] In addition, in the case of immunochromatography and the like where an immunoreaction is carried out in a solid phase, the "immunoreaction system" refers to a solid phase where a liquid sample and a binding partner undergo an immunoreaction. In this case, the sample may be pre-mixed with a pretreatment solution containing both or either of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody and then dripped onto the immunochromatography test piece, and both or either of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody may be dried and retained on the sample pad, and when the sample is dripped, both or either of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody dissolves and develops the solid phase, thereby becoming present in the reaction system.

[0099] In the present invention, the concentration of the anti-L chain λ monoclonal antibody may be any concentration that does not strongly affect the immune reaction between the analyte and the specific binding partner and can exert the desired non-specific reaction inhibitory effect, and can be appropriately set by those skilled in the art according to the type of the analyte and the sample.

[0100] The concentration of the anti-L chain λ monoclonal antibody in the immune reaction system varies depending on the reagent composition of the immune reaction system. The weight of the anti-L chain λ antibody relative to 10 μL of the sample is 0.1 to 1000 μg, preferably 1.0 to 750 μg, more preferably 2.0 to 500 μg, further preferably 3.0 to 250 μg, and most preferably 5.0 to 150 μg, but is not limited to this concentration. For example, there are preferably 5.0 to 120 μg, 5.0 to 110 μg, 5.0 to 100 μg, 5.0 to 80 μg, 5.0 to 60 μg, and 5.0 to 50 μg. In addition, as the lower limit of the range, there are preferably 10.0 μg or more, 15.0 μg or more, and 20.0 μg or more.

[0101] The concentration of the anti-L chain κ monoclonal antibody in the immune reaction system varies depending on the reagent composition of the immune reaction system. The weight of the anti-L chain κ antibody relative to 10 μL of the sample is 0.1 to 1000 μg, preferably 1.0 to 750 μg, more preferably 2.0 to 500 μg, further preferably 3.0 to 250 μg, and most preferably 5.0 to 150 μg, but is not limited to this concentration. For example, there are cases where it is preferably 5.0 to 120 μg, 5.0 to 110 μg, 5.0 to 100 μg, 5.0 to 80 μg, 5.0 to 60 μg, and 5.0 to 50 μg. In addition, as the lower limit of the range, there are cases where it is preferably 10.0 μg or more, 15.0 μg or more, and 20.0 μg or more.

[0102] In the present invention, anti-L chain λ monoclonal antibodies and anti-L chain κ monoclonal antibodies may be used alone, as a mixture of multiple anti-L chain λ monoclonal antibodies, multiple anti-L chain κ monoclonal antibodies, or an anti-L chain λ monoclonal antibody and an anti-L chain κ monoclonal antibody.

[0103] When anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody are mixed for use, the mixing ratio can be 20:1 to 1:20, preferably 10:1 to 1:10, more preferably 5:1 to 1:5, and most preferably 2:1 to 1:2, as the weight of each antibody. The normal ratio of κ chain to λ chain in the serum of healthy normal people is in the range of 0.26 to 1.65 (reference 2). In addition, the normal ratio of undissociated κ chain to λ chain in serum is about 2.0. Those skilled in the art can appropriately select the mixing ratio according to the race, disease group, age, etc. of the sample donor as the measurement object.

[0104] Alternatively, either or both of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody in a free state not bound to an insoluble carrier or the like may be used, or either or both of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody bound to an insoluble carrier may be used.

[0105] Furthermore, when both or either of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody are used in combination of multiple types, the total concentration of the multiple antibodies is preferably within the above concentration range.

[0106] When the measuring reagent of the present invention contains both or either the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody in advance, it is preferred that they are contained in the measuring reagent in advance at a concentration within the reaction system.

[0107] (Latex Turbidimetric Immunoassay (LTIA))

[0108] The LTIA method, which is one of the immunological measurement methods of the present invention, is described below. Methods for measuring a substance to be measured by the LTIA method can be roughly divided into two types.

[0109] The first method is a method in which latex particles immobilized with a specific binding partner for a substance to be measured react with the substance to be measured to form a sandwich-type immune complex, and the substance to be measured is measured based on the degree of aggregation of the latex particles accompanying the formation of the immune complex.

[0110] The second method is to add a protein or the like fixed with multiple measurement object substances or their analogs (including fragments thereof) to a reagent to make it compete with the measurement object substances in the sample, thereby inhibiting the formation of immune complexes between the measurement object substances contained in the reagent and latex particles, and measuring the measurement object substance (antigen) by the degree of inhibition of agglutination of the latex particles accompanied by the inhibition of formation of the latex particle immune complex, wherein the latex particles are latex particles fixed with specific binding partners for the measurement object substances.

[0111] The substance to be measured and the specific binding partner for the substance to be measured can be selected arbitrarily according to the purpose. For example, when the substance to be measured is an antigen, antibodies such as polyclonal antibodies, monoclonal antibodies (including recombinant antibodies and functional fragments of each antibody) can be selected as the specific binding partner for the substance to be measured. When the substance to be measured is an antibody, antigens such as natural and recombinant antigens can be selected as the specific binding partner for the substance to be measured.

[0112] The present invention can be used in any of the above methods. Specifically, the following steps can be mentioned:

[0113] (1) a step of contacting a sample containing a substance to be measured with an anti-L chain λ monoclonal antibody or an anti-L chain κ monoclonal antibody in a solution;

[0114] (2) after step (1), adding a specific binding partner for the substance to be measured;

[0115] or

[0116] (2′) after step (1), adding latex particles carrying a specific binding partner for the substance to be measured to the solution;

[0117] (3) after the step (2), a step of detecting a signal derived from a reaction between the analyte in the solution and the specific binding partner;

[0118] or

[0119] (3') After the step (2'), a step of optically detecting the degree of aggregation of the latex particles in the solution.

[0120] Here, steps (3) and (3') refer to "a step of measuring the agglutination reaction between the substance to be measured and the latex particles without a washing / separation step during or after step (2)".

[0121] As the LTIA method, the substance to be measured can be measured by optically or electrochemically observing the degree of agglutination produced. As a method of optical observation, there can be cited a method of measuring the scattered light intensity, absorbance or transmitted light intensity by an optical device (endpoint method, rate method, etc.). The measured values ​​such as absorbance obtained by measuring the sample are compared with the measured values ​​such as absorbance obtained by measuring the standard substance (a sample in which the concentration of the measured substance is known), and the concentration of the measured substance contained in the sample is calculated (quantitative value). It should be noted that the measurement of the absorbance of transmitted light or scattered light can be measured at one wavelength or at two wavelengths (based on the difference or ratio of the two wavelengths). The measurement wavelength is usually selected from 500nm to 900nm.

[0122] The determination of the determination object substance in the sample of the present invention can be carried out by a manual method or using a device such as a determination device. The determination device can be a general automatic analysis device or a dedicated determination device (dedicated machine). In addition, the determination is preferably implemented by a method performed by multiple operation steps such as a 2-step method (2-reagent method).

[0123] (latex particles loaded with specific binding partners)

[0124] The specific binding partner for the substance to be measured can be fixed and supported on the latex particles by a known method such as physical adsorption, chemical binding, or a combination of these. In the case of the physical adsorption method, it can be carried out by mixing and contacting the specific binding partner for the substance to be measured with the latex particles in a solution such as a buffer solution, or by contacting the specific binding partner for the substance to be measured dissolved in a buffer solution with a carrier, etc. In addition, when the method is performed by chemical binding, it is performed according to the known methods described in "Clinical Pathology Temporary Supplement Special Volume No. 53 for Clinical Examination - Technology and Application -" compiled by the Japanese Society of Clinical Pathology, published in 1983; "New Chemistry Experiment Lecture 1 Protein IV" compiled by the Japan Biochemical Society, published in 1991 by Tokyo Chemical Dojin, etc., by mixing and contacting the specific binding partner for the substance to be measured and the carrier with a divalent cross-linking reagent such as glutaraldehyde, carbodiimide, imidoester or maleimide, so that the various amino groups, carboxyl groups, thiol groups, aldehyde groups or hydroxyl groups of the specific binding partner for the substance to be measured and the carrier react with the above-mentioned divalent cross-linking reagent.

[0125] The synthetic polymer constituting the latex particles of the present invention is not particularly limited, and examples thereof include polystyrene, styrene-styrene sulfonate copolymers, methacrylic acid polymers, acrylic acid polymers, itaconic acid polymers, and styrene-hydrophilic carboxyl monomer copolymers, such as styrene-methacrylic acid copolymers, styrene-acrylic acid copolymers, and styrene-itaconic acid copolymers. Among them, styrene-methacrylic acid copolymers, styrene-itaconic acid copolymers, and styrene-styrene sulfonate copolymers are preferred. Styrene and styrene-(meth)acrylic acid copolymers are particularly preferred.

[0126] The specific binding partner for a specific substance carried by the latex particles is preferably multiple in order to form a sandwich. In the case where there are multiple antibody recognition sites for a specific substance, the specific binding partner can be one. For example, when the specific binding partner is a monoclonal antibody, multiple monoclonal antibodies with different recognition sites are used. In addition, for example, when the specific binding partner is a polyclonal antibody, it can be a polyclonal antibody derived from one antiserum, or it can be a polyclonal antibody derived from multiple antisera. In addition, monoclonal antibodies and polyclonal antibodies can be used in combination.

[0127] It should be noted that when treatment is required to inhibit the natural aggregation of latex particles, non-specific reactions, etc., the treatment can be carried out by bringing proteins such as bovine serum albumin (BSA), casein, gelatin, egg white albumin or its salts, surfactants or skimmed milk powder into contact with the surface of the latex particles and coating them, and the carrier can be sealed (masked).

[0128] (Reagents for immunological assays)

[0129] The immunological assay method of the present invention is carried out using an immunological assay reagent, which contains both or either of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody in addition to the main component of the immune reaction. As the main component of the reaction, a specific binding partner (other than the anti-L chain λ antibody and the anti-L chain κ antibody) contained in the assay object can be cited, and other examples include immunoassay particles, immunochromatographic test pieces, insoluble carriers such as microplates, etc.

[0130] The reagent for immunological assay of the present invention may contain a buffer, protein, peptide, amino acid, nucleic acid, lipid, phospholipid, sugar, inorganic salt, polymer compound, surfactant, other non-specific reaction inhibitor, preservative, etc., within the range that does not inhibit the non-specific reaction inhibitory effect. The pH, ionic strength, osmotic pressure, etc. of the sample are used as buffering and adjusting components, for example, acetic acid, citric acid, phosphoric acid, Tris, glycine, boric acid, carbonic acid, phthalic acid, succinic acid, maleic acid, imidazole and other buffers and Good's buffer, their sodium salts, potassium salts, calcium salts, etc. In addition, a polymer such as polyvinyl pyrrolidone and phospholipid polymers may be contained as a component to enhance the formation of aggregation.

[0131] The concentration of either or both of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody in each constituent reagent may be contained in a manner that allows the concentration to be adjusted to the concentration within the immune reaction system in the mixed state of the reagent and the sample during measurement, and varies depending on the type of reagent.

[0132] (Reagent test kit)

[0133] As for the kit of the present invention, the kit composition includes at least both or either of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody. As the kit composition, in addition to the reagents related to the immunoassay, sample diluents, sample extracts, etc. can also be cited, and the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody of the present invention can be included in one or more of them. In addition to the above, the composition of the kit can include instructions for use, sample collection tools (collection pipettes, syringes, cotton swabs, filters, etc.).

[0134] Hereinafter, the reagent configurations used in each immunoassay method will be described.

[0135] <Latex immunoturbidimetry>

[0136] An example of a reagent in the case where the immunological assay is latex immunoturbidimetry (LTIA reagent).

[0137] (1) a first reagent comprising either or both of an anti-L chain λ monoclonal antibody and an anti-L chain κ monoclonal antibody;

[0138] (2) A second reagent comprising latex particles supporting a specific binding partner for a substance to be measured.

[0139] The first reagent typically includes a buffer, and as for the concentration of both or either of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody in the buffer, it is sufficient as long as it is included in a manner that can be adjusted to the concentration of both or either of the preferred anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody in the mixed state of the reagent and the sample during the measurement, which varies according to the type of each reagent. In addition to being included in the first reagent, both or either of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody can be included in the second reagent. It should be noted that when the anti-L chain λ antibody and the anti-L chain κ antibody are used in combination, the total amount of the anti-L chain λ antibody and the anti-L chain κ antibody can be included in a manner that can be adjusted to the preferred concentration. In the latex immunoturbidimetry of the present invention, the anti-L chain λ antibody is preferably an anti-L chain λ monoclonal antibody, and the anti-L chain κ antibody is preferably an anti-L chain κ monoclonal antibody.

[0140] In the LTIA reagent of the present invention, the concentration of the anti-L chain λ monoclonal antibody contained in the first reagent is generally 1 to 2000 μg / mL, preferably 5 to 1500 μg / mL, more preferably 10 to 1000 μg / mL, further preferably 15 to 800 μg / mL, and most preferably 25 to 800 μg / mL, but is not limited to this concentration.

[0141] Furthermore, before the LTIA method is performed, when a sample diluent, a pretreatment solution, or the like is added to a sample, the anti-L chain λ antibody may be contained in the pretreatment solution, and the concentration of the anti-L chain λ antibody in the pretreatment solution is 1 to 2000 μg / mL, preferably 5 to 1500 μg / mL, more preferably 10 to 1000 μg / mL, further preferably 15 to 800 μg / mL, and most preferably 25 to 800 μg / mL, but is not limited to this concentration.

[0142] Similarly, the concentration of the anti-L chain κ monoclonal antibody contained in the first reagent is 1 to 2000 μg / mL, preferably 5 to 1500 μg / mL, more preferably 10 to 1000 μg / mL, further preferably 15 to 800 μg / mL, and most preferably 25 to 800 μg / mL, but is not limited to this concentration.

[0143] In addition, when adding sample diluent, pretreatment solution, etc. to the sample before performing the LTIA method, the anti-L chain κ antibody can be included in the pretreatment solution, and the concentration of the anti-L chain κ antibody in the pretreatment solution is 1 to 2000 μg / mL, preferably 5 to 1500 μg / mL, more preferably 10 to 1000 μg / mL, further preferably 15 to 800 μg / mL, and most preferably 25 to 800 μg / mL, and is not limited to this concentration.

[0144] It should be noted that when the anti-L chain λ antibody and the anti-L chain κ antibody are used in combination, the total amount of the anti-L chain λ antibody and the anti-L chain κ antibody is within the above concentration range.

[0145] Regarding the immunometric particles used in the present invention, in addition to the above-mentioned latex particles, as long as they can carry a specific binding partner for the analyte, known particles can be used. For example, inorganic particles such as metal colloids, silica, and carbon can also be used as the immunometric particles of the present invention.

[0146] Regarding the size of the immunometric particles, considering the optical measurement methods used (for example, turbidimetry for measuring transmitted light, nephelometry for measuring scattered light, etc.), it can be appropriately selected from the range of 0.05 to 1 μm in such a way that the desired measurement sensitivity, measurement range, etc. are obtained. It should be noted that an average particle size of 0.1 to 0.4 μm is common in optical measurements in automated analyzers, but is not limited thereto.

[0147] <ELISA method>

[0148] The ELISA method refers to a method of detecting enzyme activity by finally incorporating an antigen or antibody labeled with an enzyme into the reaction system by using various combinations of antigen-antibody reactions. Regarding the detection of enzyme activity, substrates whose absorption spectra change with the reaction are used, and examples of combinations of antigen-antibody reactions include the direct method, the indirect method, the sandwich method, the competitive method, etc.

[0149] Examples of the reagents in the case where the immunological assay method of the present invention is the sandwich ELISA method:

[0150] (a) An insoluble carrier immobilized with an antibody that reacts with the analyte;

[0151] (b) An antibody labeled with a labeling substance and reacting with the analyte.

[0152] As the insoluble carrier of (a), a plate is preferred, and the labeling substance can be appropriately selected for use. In the case of an antibody immobilized on an insoluble carrier, the substance to be measured in a solution containing a sample is captured to form a complex on the insoluble carrier. In the case of an antibody labeled with a labeling substance, the substance to be measured is combined with the captured substance to be measured and forms a sandwich with the complex. The amount of the labeling substance can be measured by a method corresponding to the labeling substance, thereby measuring the substance to be measured in the sample. In terms of specific methods such as a method for immobilizing an antibody on an insoluble carrier and a method for combining an antibody with a labeling substance, methods known to those skilled in the art can be used without particular limitation.

[0153] In the ELISA method, both or either of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody of the present invention can be present in the immune reaction system by, for example, adding to a sample diluent, a pretreatment solution, etc., or adding to a solution for antigen-antibody reaction. In the ELISA method, the anti-L chain λ antibody is preferably an anti-L chain λ monoclonal antibody, and the anti-L chain κ antibody is preferably an anti-L chain κ monoclonal antibody.

[0154] <Immunochromatography>

[0155] The reagent configuration (test piece configuration) when the immunoassay method of the present invention is an immunochromatography method will be described.

[0156] Immunochromatographic test strips;

[0157] When an antibody is used as a specific binding partner, a test piece is provided on a sheet-like insoluble carrier such as a porous membrane, and has, in order in the development direction of a solution containing the sample, "1. a sample supplying portion", "2. a portion for retaining a labeled antibody (labeled antibody retaining portion)", and "3. a portion for immobilizing an antibody for capturing a complex formed by the labeled antibody and an antibody against the substance to be measured (capturing antibody portion)".

[0158] In the immunochromatography method, at least such a test piece is included. When a sample containing a substance to be measured is added to a sample supply portion in a given amount, the sample is immersed in the label holding portion by capillary phenomenon, and the substance to be measured is combined with the labeled antibody to form a complex. As for the complex, when the membrane is unfolded and immersed in the capture antibody portion, the antibody (capture antibody) fixed on the membrane is captured to form a complex of capture antibody-substance to be measured-label antibody. And the label can be detected by any method (for example, in the case of a label that can be visualized such as colloidal gold, it is an agglutination image, and in the case of an enzyme, it is a color reaction based on the addition of a substrate), so as to detect the substance to be measured.

[0159] In the present immunochromatography, both or either of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody of the present invention are present in the reaction system, for example, by adding to a sample diluent, a pretreatment solution, etc., or by being contained in a sample supply portion, a label holding portion, and being dried and held. In the immunochromatography, the anti-L chain λ antibody is preferably an anti-L chain λ monoclonal antibody, and the anti-L chain κ antibody is preferably an anti-L chain κ monoclonal antibody.

[0160] <Chemiluminescence method>

[0161] After the magnetic particles bound to the antigen or antibody react with the substance to be measured to form a complex, the unreacted substances are removed by magnetism. A reagent containing a labeled antibody is further added, and after the unreacted substances are removed by magnetism, a luminescent reagent is added and the luminescence amount is measured. The case where an enzyme is used in the labeling is called a chemiluminescent enzyme immunoassay (CLEIA method: Chemiluminescent enzyme immunoassay). In addition, the case where a metal complex such as a ruthenium pyridine complex is used in the labeling to measure the luminescence intensity through an electrochemical reaction is called an electrochemiluminescence immunoassay (ECLIA method: Electro chemiluminescence immunoassay). In addition, the case where a chemiluminescent substance is used in the labeling is called a chemiluminescent immunoassay (CLIA method: Chemiluminescent immunoassay).

[0162] The reagents used in the case where the immunoassay method of the present invention is the CLEIA method are exemplified as follows:

[0163] (a) magnetic particles on which antibodies (or antigens) reactive with a substance to be measured are immobilized;

[0164] (b) an antibody (or antigen) which is enzyme-labeled and reacts with a substance to be measured;

[0165] (c) Luminescent reagent.

[0166] In the case of antibodies immobilized on magnetic particles, the substance to be measured in the solution containing the sample is captured to form a complex. In the case of antibodies labeled with enzyme-labeled substances, the captured substance to be measured is bound to form a sandwich with the complex. The substance to be measured in the sample can be measured by reacting the enzyme-labeled substance with a luminescent reagent to measure the amount of luminescence.

[0167] In the CLEIA method, both or either of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody of the present invention can be present in the immune reaction system by, for example, adding to a sample diluent, a pretreatment solution, etc., or adding to a solution for antigen-antibody reaction. In the chemiluminescence method, the anti-L chain λ antibody is preferably an anti-L chain λ monoclonal antibody, and the anti-L chain κ antibody is preferably an anti-L chain κ monoclonal antibody.

[0168] <Specific Binding Partner>

[0169] In the present invention, examples of specific binding partners for the substance to be measured include proteins, peptides, amino acids, lipids, carbohydrates, nucleic acids, haptens, and the like other than anti-L chain λ monoclonal antibodies and anti-L chain κ monoclonal antibodies. There are no particular limitations on the molecular weight or the origin such as natural or synthetic. Examples include antibodies or antigens that can be used in immunological assays utilizing immune reactions.

[0170] The antibody may be a polyclonal antibody or a monoclonal antibody, more preferably a monoclonal antibody.

[0171] As the antibody of the present invention, in addition to the whole antibody molecule, the functional fragment of the antibody with antigen-antibody reactivity can also be used. In addition to obtaining by immunizing common animals (mouse, goat, sheep, etc.), it can be an antibody (chimeric antibody, humanized antibody or fully human antibody, etc.) in which the amino acid sequence of an animal species different from the animal of the immune immunogen (substance to be measured) is modified by gene recombination technology, etc. As the functional fragment of the antibody, F (ab') 2, Fab', single-chain antibody (scFv), VHH (variable domain of heavy chain of heavy chain antibody) antibody, IgNAR (newantigenreceptor) antibody, etc., which are fragments with antigen-antibody reactivity, can be cited. The functional fragments of these antibodies can be manufactured by treating the antibodies obtained as described above with protease (e.g., pepsin, papain, etc.).

[0172] <Sample>

[0173] Examples of the sample containing the substance to be measured in the present invention include human or animal blood, serum, plasma, culture supernatant, urine, spinal fluid, saliva, sweat, ascites, or cell or tissue extracts.

[0174] In addition, the samples in the present invention include not only the samples obtained from the organism itself, but also samples that have been pre-treated by dilution, purification, etc. In addition, the samples in the present invention may be any of those that have just been collected or those that have passed a certain time after collection. The present invention has an inhibitory effect on non-specific factors that still exist after a certain time after collection.

[0175] In addition, it also has an inhibitory effect on non-specific factors present in samples that have been frozen and stored for a certain period of time and then thawed, or samples that have been repeatedly frozen and thawed. The certain period of time may be immediately after collection, one day, one week, one month, etc.

[0176] <Measurement target substance>

[0177] The immunoassay reagent of the present invention can use various substances contained in the sample as the assay target substance. The assay target substance is a substance that does not react with the anti-L chain λ antibody and the anti-L chain κ antibody used for non-specific reaction suppression, that is, it can be any substance other than the immunoglobulin L chain λ chain and the immunoglobulin L chain κ chain. Proteins, peptides, amino acids, lipids, carbohydrates, nucleic acids, haptens, etc. other than the above can be cited, and there is no particular limitation as long as it is a substance that can be measured in theory. For example, 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 fibrinogen), TAT (thrombin-antithrombin III complex), PAI-1, phenytoin, phenobarbital, carbamazepine, valproic acid, theophylline, TARC (thymus and activation-regulated chemokine), sIL-2R (soluble interleukin-2 receptor), SP-D (pulmonary surfactant protein D), etc. can be mentioned.

[0178] However, the measurement target substance does not include anti-immunoglobulin L chain λ antibody and anti-immunoglobulin L chain κ antibody. It should be noted that the anti-immunoglobulin L chain λ antibody and anti-immunoglobulin L chain κ antibody mentioned here are not limited to monoclonal antibodies, but also include polyclonal antibodies.

[0179] <Composition and non-specific reaction inhibitor>

[0180] Furthermore, the present invention provides a composition comprising both or either of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody.

[0181] The composition can be used in immunological assays as a non-specific reaction inhibitor comprising, for example, both or either of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody as active ingredients.

[0182] In the present invention, inhibiting non-specific reaction means that the non-specific reaction inhibitor plays a role in the factor (non-specific factor) that produces the non-specific reaction in the biological sample, and inhibits the influence of the reaction other than the immune reaction between the measured object substance and the specific binding partner on the determination. Therefore, in the present invention, as for whether the candidate substance as a non-specific reaction inhibitor has an inhibitory effect on non-specific reaction, for example, the measured value (hereinafter referred to as the control method measured value) can be used as a benchmark by a method that does not produce non-specific reaction in a certain sample (or, a method that is not easy to produce (in the embodiment, CLEIA method)) The case where the candidate substance is added is compared with the case where it is not added, and it is judged according to whether it is close to the control method measured value. That is, in the target determination method, the measured value of the case where the candidate substance is added is compared with the measured value of the case where the candidate substance is not added. In the case of being close to the control method measured value, it is judged that the candidate substance has a non-specific reaction inhibitory effect in the determination method and is a non-specific reaction inhibitor.

[0183] The composition and non-specific reaction inhibitor in the present invention target the following factors: factors that generate so-called positive measurement errors due to a certain component contained in a biological sample, and factors that generate so-called negative measurement errors due to a value that determines the original value to be lower. Among them, it is particularly effective for non-specific factors that cannot be inhibited even by commercially available non-specific reaction inhibitors such as HBR-1. In addition, it is effective for non-specific factors contained in so-called deviation samples, which generate positive measurement errors in which the measurement value is abnormally high, and negative measurement errors in which the measurement value is abnormally low.

[0184] The composition and non-specific reaction inhibitor of the present invention may contain a substance capable of inhibiting a reaction based on a non-specific factor derived from a sample as determined above, and may contain at least an anti-L chain λ monoclonal antibody or an anti-L chain κ monoclonal antibody as an active ingredient. The non-specific reaction inhibitor of the present invention may use the composition containing the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody, the anti-L chain λ monoclonal antibody or the anti-L chain κ monoclonal antibody in the above-mentioned reagent composition as it is.

[0185] The nonspecific reaction inhibitor of the present invention may include buffers, proteins, peptides, amino acids, nucleic acids, lipids, phospholipids, sugars, inorganic salts, polymer compounds, surfactants, other nonspecific reaction inhibitors (anti-C3d antibodies, anti-human IgM antibodies, etc.), preservatives, etc., within the range that does not inhibit the nonspecific reaction inhibitory effect.

[0186] <Methods for inhibiting non-specific reactions>

[0187] The method for suppressing nonspecific reactions of the present invention is a method for suppressing nonspecific reactions caused by a sample by causing an immune reaction in the presence of an anti-L chain λ monoclonal antibody and an anti-L chain κ monoclonal antibody, in the presence of an anti-L chain λ monoclonal antibody, or in the presence of an anti-L chain κ monoclonal antibody.

[0188] [References]

[0189] 1: Edward A. Greenfield (Ed.) (2012) Antibodies: ALaboratory Manual (Second Edition), Cold Spring Harbor Laboratory Press, 750pp.

[0190] 2: Jerry A.Katzmann, Raynell J.Clark, Roshini S.Abraham, Sandra Bryant, James F.Lymp, Arthur R.Bradwell, and Robert A.Kyle (2002) Serum ReferenceIntervals and Diagnostic Ranges for Freeκand FreeλImmunoglobulin LightChains: Relative Sensitivity for Detection of Monoclonal Light Chains,ClinicalChemistry,48(9):1437-1444.

[0191] Example

[0192] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to the following examples.

[0193] [Reference Example 1] (Production of human IgA immune-derived monoclonal antibodies)

[0194] 1. Preparation of hybridomas from human IgA-immunized animals

[0195] (1) Immunization of animals

[0196] As an immunogen, commercially available human IgA (manufactured by BETHYL Laboratories) was used. The immunogen was diluted with 20 mM phosphate buffer (pH 7.2; hereinafter referred to as PBS) containing 150 mM sodium chloride, and the immunogen was mixed with Freund's complete adjuvant in equal amounts to prepare an emulsion, and 30 μg was injected into each Balb / cAJcl mouse using the emulsion. In addition, Freund's incomplete adjuvant was used after the second time, 20 μg per injection, and the injection was repeated 3 to 5 times at intervals of 7 to 10 days. The antibody titer in the antiserum obtained by tail vein blood collection was measured by the antigen solid-phase ELISA method described below.

[0197] (2) Evaluation of antibody titer in immunized animal serum (antigen solid-phase ELISA method)

[0198] The presence of antibodies against human IgA in the serum of the immunized animals was confirmed by ELISA method in which the immunogen was immobilized on a solid phase (antigen immobilized ELISA method). The details of the antigen immobilized ELISA method are as follows.

[0199] (2-1) Preparation of antigen-immobilized ELISA plate

[0200] Human IgA as an immunogen was dissolved in PBS to a concentration of 1.0 μg / mL, and 50 μL of the resulting solution was dispensed into each well of a 96-well microplate and allowed to stand at room temperature for 2 hours.

[0201] 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 100 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.

[0202] (2-2) Antigen solid phase ELISA method

[0203] After removing BSA-PBST from each well of the ELISA plate, 50 μL of immune animal antiserum and non-immune animal serum obtained by step dilution with BSA-PBST were added to each well and left to stand at room temperature for 1 hour. After washing each well 3 times with 400 μL of PBST, HRP-labeled anti-mouse IgG (H&L) (manufactured by Southern Biotechnology Associates Inc.) obtained by diluting 10,000 times with BSA-PBST was dispensed into each well with 50 μL and left to stand at room temperature for 1 hour. After washing each well 3 times with 400 μL of PBST, 50 μL of citric acid buffer (pH 5.0) containing 0.2% o-phenylenediamine and 0.02% hydrogen peroxide was added, and after standing at room temperature for 10 minutes, 50 μL of 1.5N sulfuric acid was added to stop the enzyme reaction, and the absorbance at a wavelength of 492 nm was measured. As a result of the measurement, spleens and lymph nodes were removed from mice in which an increase in antibody titer was confirmed, and spleen- and lymph node-derived cells were prepared and used for cell fusion.

[0204] (3) Cell fusion

[0205] The spleen-derived cells or lymph node-derived cells were mixed with myeloma cells at a ratio of 6 to 1, and cell fusion was performed by the PEG method. The cells obtained by cell fusion were suspended in HAT medium and cultured in a CO2 incubator at 37°C and 5% CO2 for 8 days to obtain fused cells (hybridomas).

[0206] 2. Selection of hybridomas (antigen solid phase ELISA method)

[0207] The presence of antibodies against IgA in the culture supernatant of the hybridoma was confirmed by ELISA in which the immunogen was immobilized (antigen immobilized ELISA method). The details of the antigen immobilized ELISA method are as follows.

[0208] (1) Preparation of antigen-immobilized ELISA plates

[0209] IgA as an immunogen was dissolved in PBS to a concentration of 1.0 μg / mL, and 50 μL of the obtained solution was dispensed into each well of a 96-well microplate and allowed to stand at room temperature for 2 hours.

[0210] Each well was washed three times with 400 μL of PBST, and then 100 μL of BSA-PBST was added and blocked at room temperature for 1 hour. This was used as an ELISA plate.

[0211] (2) Antigen solid-phase ELISA method

[0212] After removing BSA-PBST from each well of the ELISA plate, 50 μL of the culture supernatant of the hybridoma obtained by step dilution with BSA-PBST was added to each well and left to stand at room temperature for 1 hour. After washing each well with PBST400 μL 3 times, HRP-labeled anti-mouse IgG (H&L) diluted 10,000 times with BSA-PBST was dispensed into each well with 50 μL and left to stand at room temperature for 1 hour. After washing each well with PBST400 μL 3 times, 50 μL of citric acid buffer (pH 5.0) containing 0.2% o-phenylenediamine and 0.02% hydrogen peroxide was added, and after standing at room temperature for 10 minutes, 50 μL of 1.5N sulfuric acid was added to stop the enzyme reaction, and the absorbance at a wavelength of 492 nm was measured.

[0213] As a result of the measurement, wells with higher absorbance were selected as wells in which anti-human IgA antibody-producing hybridomas were present (positive wells).

[0214] 3. Cloning

[0215] Using the anti-human IgA antibody-producing hybridoma selected by 2. above, single cloning of the hybridoma and purification of the monoclonal antibody were performed. Cloning was performed by the limiting dilution method, and positive wells were selected by the same method as the above-mentioned antigen solid phase ELISA method, and finally a plurality of monoclonal antibody-producing hybridomas were obtained. The monoclonal antibodies produced by the obtained hybridoma S062XX (XX is a number) are respectively referred to as S062XX antibodies.

[0216] [Reference Example 2] (Evaluation of specificity of human IgA immune-derived monoclonal antibodies)

[0217] 1. Specificity evaluation of human IgA immune-derived monoclonal antibodies (antigen solid phase ELISA method)

[0218] The specificity of the human IgA immunization-derived monoclonal antibody was confirmed by an ELISA method in which the antigen was immobilized (antigen immobilized ELISA method). The details of the antigen immobilized ELISA method are as follows.

[0219] (1) Preparation of antigen-immobilized ELISA plates

[0220] Antigens for specificity evaluation (IgG Kappa, IgG Lambda (both manufactured by Bethyl Laboratories Inc.), IgA Kappa, IgA Lambda (both manufactured by Southern Biotechnology Associates Inc.) were dissolved in PBS to 1.0 μg / mL, and 50 μL of the obtained solution was dispensed into each well of a 96-well microplate and allowed to stand at room temperature for 2 hours.

[0221] Each well was washed three times with 400 μL of PBST, and then 100 μL of BSA-PBST was added and blocked at room temperature for 1 hour. This was used as an ELISA plate.

[0222] (2) Antigen solid-phase ELISA method

[0223] After removing BSA-PBST from each well of the ELISA plate, 50 μL of the antibody diluted to 1.0 μg / mL and 0.2 μg / mL with BSA-PBST was added to each well and left to stand at room temperature for 1 hour. After washing each well 3 times with PBST400 μL, HRP-labeled anti-mouse IgG (H&L) diluted 9500 times with BSA-PBST was dispensed into each well with 50 μL and left to stand at room temperature for 1 hour. After washing each well 3 times with PBST400 μL, 50 μL of citric acid buffer (pH 5.0) containing 0.2% o-phenylenediamine and 0.02% hydrogen peroxide was added. After standing at room temperature for 10 minutes, 50 μL of 1.5N sulfuric acid was added to stop the enzyme reaction and the absorbance at a wavelength of 492 nm was measured. The results of the measurement are shown in Table 1.

[0224] 2. Specificity evaluation results

[0225] Among the multiple human IgA immune-derived monoclonal antibodies, it was confirmed that: specific antibodies for IgA (S06201, S06216) react with IgA Kappa and IgA Lambda, and do not react with IgG Kappa and IgG Lambda; specific antibodies for L chain κ (S06205, S06204), which react with IgG Kappa and IgA Kappa, and do not react with IgG Lambda and IgALambda; specific antibodies for L chain λ (S06215, S06208), which react with IgG Lambda and IgA Lambda, and do not react with IgG Kappa and IgA Kappa. It should be noted that the reason for judging it as the specific antibody for IgA is as follows. That is, in terms of the reaction with the two antigens of the κ chain and the λ chain of IgA, it is because it is an antibody that reacts with the H chain, not an antibody that reacts with the L chain. Furthermore, since the antibody does not react with the λ chain and κ chain of IgG, it is considered to be an IgA-specific antibody.

[0226] Thus, the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody of the present invention were selected from monoclonal antibodies derived from human IgA immunity, and their effect of suppressing nonspecific reactions in immunoassays was evaluated.

[0227] [Table 1]

[0228] Evaluation of the specificity of monoclonal antibodies derived from human IgA immunity

[0229]

[0230] Abs.(OD)at 492nm

[0231] [Experimental Example 1: Inhibition of non-specific reactions in the LTIA method: Measurement of sIL-2R]

[0232] The following test is shown: using a reagent based on the LTIA method, the reagent is added with the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody of the present invention, and other non-specific reaction inhibitors, and the sIL-2R concentration in the sample is measured. It should be noted that the following samples were used as samples: samples showing values ​​close to the measurement values ​​based on the chemiluminescent enzyme immunoassay (CLEIA method) (control samples: samples 1 to 6); and samples showing non-specific reactions and having a large deviation from the measurement values ​​of the control example 1 (deviation samples: samples 7 to 11).

[0233] [Comparative Example 1] (Measurement based on the CLEIA method)

[0234] 1. Determination method

[0235] 1-1. Assay reagents

[0236] LUMIPULSE PRESTO (registered trademark) IL-2R (FUJIREBIO CORPORATION)

[0237] 1-2. Sample

[0238] Sample (serum) 1-11

[0239] 1-3. Measurement steps

[0240] The measurement was performed using LUMIPULSE (registered trademark)-L2400 (Fujire Bio Co., Ltd.) according to the instruction manual of the measurement reagent.

[0241] 2. Measurement results

[0242] The measurement results are shown in Table 2.

[0243] The CLEIA method described in this comparative example 1 carries out a B / F separation operation and has a washing step, so it is used as a comparative example because it is a measurement method that is not easily affected by non-specific reactions derived from samples.

[0244] [Comparative Example 1] (Measurement by LTIA method: without adding non-specific reaction inhibitor)

[0245] 1. Determination method

[0246] 1-1. Assay reagents

[0247] According to the method described in Japanese Patent Application Laid-Open No. 2017-181377, the first reagent (buffer) and the second reagent (anti-sIL-2R antibody-sensitized latex particle solution) described below were prepared.

[0248] 1-2. Sample

[0249] Same as the sample of Control 1.

[0250] 1-3. Measurement steps

[0251] The first reagent and the second reagent were combined and the sIL-2R concentration in the sample was measured using a Hitachi 7180 automatic analyzer. Specifically, 120 μL of the first reagent was added to 5.6 μL of the sample and kept at 37°C for 5 minutes, and then 40 μL of the second reagent was added and stirred. After 5 minutes, the absorbance change accompanying the formation of agglutination was measured at a main wavelength of 570 nm and a sub-wavelength of 800 nm, and the absorbance change was fitted to a calibration curve obtained by measuring a standard substance with a known concentration, and the measured value was calculated.

[0252] 2. Measurement results

[0253] The measurement results are shown in Table 2.

[0254] [Comparative Example 2] (Measurement by LTIA method: Addition of HBR-1)

[0255] The measurement was performed in the same manner as in Comparative Example 1, except that HBR-1 (manufactured by SCANTIBODIES LABORATORY, INC.), which is a commercially available nonspecific reaction inhibitor, was added to the first reagent in Comparative Example 1 so as to have a concentration of 200 μg / mL.

[0256] [Comparative Example 3] (Measurement based on the LTIA method: Addition of anti-human IgG, IgA, and IgM polyclonal antibodies)

[0257] The measurement was performed in the same manner as in Comparative Example 1 except that commercially available anti-human IgG polyclonal antibody, anti-human IgA polyclonal antibody, and anti-human IgM polyclonal antibody (manufactured by SeraCare Life Sciences, Inc. S) were added to the first reagent in Comparative Example 1 to a concentration of 200 μg / mL. The measurement results are shown in Table 2.

[0258] [Example 1] (Measurement based on LTIA method: adding anti-L chain λ monoclonal antibody)

[0259] The measurement was performed in the same manner as in Comparative Example 1, except that anti-L chain λ monoclonal antibody (S06215 antibody prepared in Reference Example 1) was added to the first reagent in Comparative Example 1 to a concentration of 200 μg / mL.

[0260] [Example 2] (Measurement based on LTIA method: adding anti-L chain κ monoclonal antibody)

[0261] The measurement was performed in the same manner as in Comparative Example 1, except that the anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) was added to the first reagent in Comparative Example 1 to a concentration of 200 μg / mL.

[0262] [Example 3] (Measurement based on LTIA method: adding anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody)

[0263] The measurement was performed in the same manner as in Comparative Example 1, except that the anti-L chain λ monoclonal antibody (S06215 antibody prepared in Reference Example 1) was added to the first reagent as in Comparative Example 1 so as to be 200 μg / mL, and the anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) was added to be 200 μg / mL. The measurement results are shown in Table 2.

[0264] [Table 2]

[0265] sIL-2R measurement results (U / mL)

[0266]

[0267] <Results and Investigation>

[0268] Based on the results of Control Example 1, Comparative Examples 1 to 3, and Examples 1 to 3, the effects of the present invention were examined.

[0269] (1) The measurement results of the control samples (sample numbers 1 to 6) were verified.

[0270] In the control sample, the measured value of the CLEIA method (Control Example 1) is roughly the same as the measured value of the LTIA method (Comparative Example 1) without adding a non-specific reaction inhibitor. In addition, the measured value of the LTIA method (Example 1) added with the anti-L chain λ monoclonal antibody of the present invention, the measured value of the LTIA method (Example 2) added with the anti-L chain κ monoclonal antibody, and the measured value of the LTIA method (Example 3) using the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody in combination are also roughly the same as the control example 1 and the comparative example 1. Therefore, it can be seen that the addition of anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody has no effect on the measured value of the sample that does not show a non-specific reaction. On the other hand, it was confirmed that the measured value of the LTIA method (Comparative Example 3) added with anti-human IgG polyclonal antibody, anti-human IgA polyclonal antibody and anti-human IgM polyclonal antibody has a tendency to further deviate compared with the comparative example 1.

[0271] (2) The measurement results of the deviation samples (sample numbers 7 to 11) were verified.

[0272] In sample number 7, the measured value of the CLEIA method (Control Example 1) was 672 U / mL, while the measured value of the LTIA method (Comparative Example 1) without adding a non-specific reaction inhibitor was 1814 U / mL, and the measured value deviation was large. In addition, the measured value of the LTIA method with the addition of HBR-1 (Comparative Example 2) was 2037 U / mL, which was significantly deviated from Control Example 1. The measured value of the LTIA method (Comparative Example 3) with the addition of anti-human IgG polyclonal antibody, anti-human IgA polyclonal antibody, and anti-human IgM polyclonal antibody was 1060 U / mL, confirming the presence of a non-specific reaction inhibitory effect, but since there was a deviation from Control Example 1, the non-specific reaction inhibitory effect based on this substance was insufficient.

[0273] On the other hand, the measured value by the LTIA method (Example 1) in which the anti-L chain λ monoclonal antibody of the present invention was added was 587 U / mL, and the measured value by the LTIA method (Example 3) in which the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody were used in combination was 617 U / mL, showing a tendency close to that of Control Example 1. The same results were obtained in Samples 8 and 9.

[0274] In addition, in sample number 10, the measured value of the CLEIA method (Control Example 1) was 1640 U / mL, while the measured value of the LTIA method without adding a non-specific reaction inhibitor (Comparative Example 1) was 1857 U / mL, and there was a deviation in the measured value. The measured value of the LTIA method with the addition of HBR-1 (Comparative Example 2) was 1883 U / mL, which was significantly different from that of Control Example 1. The measured value of the LTIA method with the addition of anti-human IgG polyclonal antibody, anti-human IgA polyclonal antibody, and anti-human IgM polyclonal antibody (Comparative Example 3) was 2385 U / mL, and the degree of deviation was greater than that of Control Example 1.

[0275] On the other hand, the measured value of the LTIA method of the present invention with the addition of anti-L chain κ monoclonal antibody (Example 2) was 1529 U / mL, and the measured value of the LTIA method using a combination of anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody (Example 3) was 1567 U / mL, showing a tendency close to that of Control Example 1. The same result was obtained in Sample 11.

[0276] (3) As described above, in the immunoassay method, nonspecific reactions derived from the sample can be suppressed by allowing the anti-L chain λ monoclonal antibody or anti-L chain κ monoclonal antibody of the present invention to be present in the immune reaction system.

[0277] According to the present invention, non-specific reactions that cannot be inhibited by HBR-1, anti-human IgG polyclonal antibody, anti-human IgA polyclonal antibody, and anti-human IgM polyclonal antibody, which are commercially available non-specific reaction inhibitors, can be inhibited. The non-specific reaction inhibitory effect of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody of the present invention is an unexpected result. In addition, various non-specific reactions derived from samples can be inhibited by using the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody in combination.

[0278] It should be noted that the anti-L chain λ monoclonal antibody alone in sample numbers 7 to 9 also has a non-specific reaction inhibitory effect. The anti-L chain κ monoclonal antibody alone in sample numbers 10 and 11 also has a non-specific reaction inhibitory effect. In this way, the anti-L chain λ monoclonal antibody or the anti-L chain κ monoclonal antibody shows that it has an effect of inhibiting non-specific reactions for a specific sample alone. On the other hand, the effect varies depending on the sample, so it is preferred to use the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody in combination.

[0279] [Experimental Example 2: Non-specific reaction inhibition in LTIA method: measurement of SP-D]

[0280] The following test is shown: using a reagent based on the LTIA method, the reagent is added with the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody of the present invention, and other non-specific reaction inhibitors, and the SP-D concentration in the sample is measured. It should be noted that the following samples were used as samples: samples showing values ​​similar to the measurement values ​​based on the chemiluminescent enzyme immunoassay (CLEIA method) (control samples: samples 12 to 15); and samples showing non-specific reactions and having a large deviation from the measurement values ​​of Control Example 2 (deviation samples: samples 16 and 17).

[0281] [Comparative Example 2] (Measurement based on the CLEIA method)

[0282] 1. Determination method

[0283] 1-1. Assay reagents

[0284] CL SP-D "YAMASA" NX (YAMASA Soy Sauce Co., Ltd.)

[0285] 1-2. Sample

[0286] Samples (serum) 12~17

[0287] 1-3. Measurement steps

[0288] The measurement was performed using CL-JACK NX (MINARIS MEDICAL CO., LTD.) according to the instruction manual of the measurement reagent.

[0289] 2. Measurement results

[0290] The measurement results are shown in Table 3.

[0291] The CLEIA method described in this comparative example 2 carries out a B / F separation operation and has a washing step, so it is used as a comparative example because it is a measurement method that is not easily affected by non-specific reactions derived from samples.

[0292] [Comparative Example 4] (Measurement by LTIA method: without adding non-specific reaction inhibitor)

[0293] 1. Determination method

[0294] 1-1. Assay reagents

[0295] The first and second reagents described below were prepared.

[0296] (1) Reagent No. 1

[0297] 100 mM MES-NaOH (pH 6.0)

[0298] 500mM NaCl

[0299] 0.5% BSA

[0300] Sensitizer

[0301] (2) Second reagent

[0302] Anti-human SP-D monoclonal antibody sensitized latex

[0303] 5mM MOPS-NaOH (pH 7.0)

[0304] 1-2. Sample

[0305] Same as the sample of Control 2.

[0306] 1-3. Measurement steps

[0307] The first reagent and the second reagent were combined and the SP-D concentration in the sample was measured using a Hitachi 7180 automatic analyzer. Specifically, 120 μL of the first reagent was added to 5.0 μL of the sample and kept at 37°C for 5 minutes, and then 40 μL of the second reagent was added and stirred. After 5 minutes, the absorbance change accompanying the formation of agglutination was measured at a main wavelength of 570 nm and a sub-wavelength of 800 nm, and the absorbance change was fitted to the calibration curve obtained by measuring a standard substance with a known concentration, and the measured value was calculated.

[0308] 2. Measurement results

[0309] The measurement results are shown in Table 3.

[0310] [Comparative Example 5] (Measurement by LTIA method: Addition of HBR-1)

[0311] The measurement was performed in the same manner as in Comparative Example 4, except that HBR-1 (SCANTIBODIES LABORATORY, INC.), a commercially available nonspecific reaction inhibitor, was added to the first reagent in Comparative Example 4 to a concentration of 200 μg / mL. The measurement results are shown in Table 3.

[0312] [Example 4] (Measurement based on LTIA method: adding anti-L chain λ monoclonal antibody)

[0313] The measurement was performed in the same manner as in Comparative Example 4 except that the anti-L chain λ monoclonal antibody (S06215 antibody prepared in Reference Example 1) was added to the first reagent in Comparative Example 4 to a concentration of 200 μg / mL.

[0314] [Example 5] (Measurement based on LTIA method: adding anti-L chain κ monoclonal antibody)

[0315] The measurement was performed in the same manner as in Comparative Example 4, except that the anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) was added to the first reagent in Comparative Example 4 to a concentration of 200 μg / mL. The measurement results are shown in Table 3.

[0316] [Example 6] (Measurement based on LTIA method: adding anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody)

[0317] The measurement was performed in the same manner as in Comparative Example 4, except that the anti-L chain λ monoclonal antibody (S06215 antibody prepared in Reference Example 1) was added to the first reagent as in Comparative Example 4 so as to be 200 μg / mL, and the anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) was added to be 200 μg / mL. The measurement results are shown in Table 3.

[0318] [Table 3]

[0319] SP-D measurement results (ng / mL)

[0320]

[0321] <Results and Investigation>

[0322] Based on the results of Control Example 2, Comparative Examples 4 to 5, and Examples 4 to 6, the effects of the present invention were examined.

[0323] (1) The measurement results of the control samples (sample numbers 12 to 15) were verified.

[0324] In the control sample, the measured value of the CLEIA method (Control Example 2) was substantially the same as the measured value of the LTIA method (Comparative Example 4) without adding a non-specific reaction inhibitor. In addition, the measured value of the LTIA method (Example 4) with the addition of the anti-L chain λ monoclonal antibody of the present invention, the measured value of the LTIA method (Example 5) with the addition of the anti-L chain κ monoclonal antibody, and the measured value of the LTIA method (Example 6) using a combination of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody were also substantially the same as those of Control Example 2 and Comparative Example 4. Therefore, it can be seen that the addition of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody has no effect on the measured value of the sample that does not show a non-specific reaction.

[0325] (2) The measurement results of the deviation samples (sample numbers 16 and 17) were verified.

[0326] In sample number 16, the measured value by the CLEIA method (Control Example 2) was 15.0 ng / mL, while the measured value by the LTIA method without adding a non-specific reaction inhibitor (Comparative Example 4) was 29.0 ng / mL, indicating a large deviation in the measured values. In addition, the measured value by the LTIA method with the addition of HBR-1 (Comparative Example 5) was 25.3 ng / mL, which was a large deviation from Control Example 2.

[0327] On the other hand, the measured value of the LTIA method with the anti-L chain λ monoclonal antibody of the present invention added (Example 4) was 13.6 ng / mL, and the measured value of the LTIA method using the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody in combination (Example 6) was 14.1 ng / mL, showing a tendency close to Comparative Example 5.

[0328] In addition, in sample number 17, the measured value of the CLEIA method (Control Example 2) was 15.0 ng / mL, while the measured value of the LTIA method (Comparative Example 4) without adding a non-specific reaction inhibitor was 37.9 ng / mL, and the measured value deviation was large. The measured value of the LTIA method with the addition of HBR-1 (Comparative Example 5) was 17.2 ng / mL, showing a tendency close to Comparative Example 5. The measured value of the LTIA method with the addition of the anti-L chain λ monoclonal antibody of the present invention (Example 4) was 27.2 ng / mL, and the measured value of the LTIA method with the addition of the anti-L chain κ monoclonal antibody (Example 5) was 27.8 ng / mL, and the degree of deviation relative to Comparative Example 4 was reduced. In addition, the measured value of the LTIA method (Example 6) using a combination of anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody was 22.0 ng / mL, showing a tendency close to Comparative Example 4 compared to the measured values ​​of Examples 4 and 5.

[0329] (3) As described above, in the immunoassay method, nonspecific reactions derived from the sample can be suppressed by allowing the anti-L chain λ monoclonal antibody or anti-L chain κ monoclonal antibody of the present invention to be present in the immune reaction system. According to the present invention, nonspecific reactions that cannot be suppressed by HBR-1, which is a commercially available nonspecific reaction inhibitor, can be suppressed, so the nonspecific inhibitory effect of the anti-L chain λ monoclonal antibody or anti-L chain κ monoclonal antibody of the present invention is an unexpected result. In addition, by using the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody in combination, an additive effect of suppressing nonspecific reactions was confirmed.

[0330] [Experimental Example 3: Inhibition of non-specific reactions in the LTIA method: Measurement of TARC]

[0331] The following test is shown: using a reagent based on the LTIA method, the reagent is added with the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody of the present invention, and other non-specific reaction inhibitors, and the TARC concentration in the sample is measured. It should be noted that the following samples were used as samples: samples showing values ​​close to the measured values ​​(Control Example 3) based on the chemiluminescent enzyme immunoassay (CLEIA method) (Control samples: Samples 18 to 23); and samples showing non-specific reactions and having a large deviation from the measured values ​​of Control Example 3 (Deviation samples: Samples 24 to 30).

[0332] [Comparative Example 3] (Measurement based on CLEIA method)

[0333] 1. Determination method

[0334] 1-1. Assay reagents

[0335] HISCL (registered trademark) TARC (SYSMEX Co., Ltd.)

[0336] 1-2. Sample

[0337] Sample (serum) 18~30

[0338] 1-3. Measurement steps

[0339] The measurement was performed using HISCL (registered trademark)-5000 (SYSMEX Corporation) according to the instructions of the reagent.

[0340] 2. Measurement results

[0341] The measurement results are shown in Table 4.

[0342] The CLEIA method described in this comparative example 3 carries out a B / F separation operation and has a washing step, so it is used as a comparative example because it is a measurement method that is not easily affected by non-specific reactions derived from samples.

[0343] [Comparative Example 6] (Measurement by LTIA method)

[0344] 1. Determination method

[0345] 1-1. Assay reagents

[0346] According to the method described in WO2022 / 009974, an LTIA assay reagent comprising a first reagent (buffer) and a second reagent (anti-human TARC monoclonal antibody sensitized latex solution) was prepared. It should be noted that HeteroBlock (manufactured by OMEGA Biologicals, Inc.), a commercially available non-specific reaction inhibitor, was added to the first reagent at a concentration of 100 μg / mL.

[0347] 1-2. Sample

[0348] Same as the sample of control example 3

[0349] 1-3. Measurement steps

[0350] The first reagent and the second reagent were combined, and the TARC concentration in the sample was measured using the automatic analyzer 3500. Specifically, 120 μL of the first reagent was added to 2.4 μL of the sample and kept at 37°C for 5 minutes, and then 40 μL of the second reagent was added and stirred. After 5 minutes, the absorbance change accompanying the formation of agglutination was measured at a main wavelength of 570 nm and a sub-wavelength of 800 nm, and the absorbance change was fitted to the calibration curve obtained by measuring a standard substance with a known concentration, and the measured value was calculated.

[0351] 2. Measurement results

[0352] The measurement results are shown in Table 4.

[0353] [Example 7] (Measurement based on LTIA method: adding anti-L chain λ monoclonal antibody)

[0354] The measurement was performed in the same manner as in Comparative Example 6, except that the anti-L chain λ monoclonal antibody (S06215 antibody prepared in Reference Example 1) was added to the first reagent shown in Comparative Example 6 so as to have a concentration of 50 μg / mL.

[0355] [Example 8] (Measurement based on LTIA method: adding anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody)

[0356] The measurement was performed in the same manner as in Comparative Example 6, except that 50 μg / mL of anti-L chain λ monoclonal antibody (S06215 antibody prepared in Reference Example 1) and anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) were added to the first reagent shown in Comparative Example 6. The measurement results are shown in Table 4.

[0357] [Table 4]

[0358] TARC measurement results (pg / mL)

[0359]

[0360] <Results and Investigation>

[0361] Based on the results of Control Example 3, Comparative Example 6, Example 7 and Example 8, the effects of the present invention were investigated.

[0362] (1) The measurement results of the control samples (sample numbers 18 to 23) were verified.

[0363] In the control sample, the measured value of the CLEIA method (Control Example 3) was substantially the same as the measured value of the LTIA method (Comparative Example 6) to which the commercially available non-specific reaction inhibitor HeteroBlock was added. In addition, the measured value of the LTIA method (Example 7) to which the anti-L chain λ monoclonal antibody of the present invention was added, and the measured value of the LTIA method (Example 8) using the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody in combination were substantially the same as those of Control Example 3 and Comparative Example 6. Therefore, it was found that the addition of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody had no effect on the measured value of the sample that did not exhibit a non-specific reaction.

[0364] (2) The measurement results of the deviation samples (sample numbers 24 to 30) were verified.

[0365] In sample No. 24, the measured value by the CLEIA method (Control Example 3) was 390 pg / mL, while the measured value by the LTIA method with the addition of HeteroBlock (Comparative Example 6) was 737 pg / mL, indicating a large deviation in the measured values.

[0366] On the other hand, the measured value of the LTIA method in which the anti-L chain λ monoclonal antibody of the present invention was added (Example 7) was 444 pg / mL, and the measured value of the LTIA method in which the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody were used in combination (Example 8) was 428 pg / mL, showing a tendency close to that of Control Example 3.

[0367] In addition, in sample number 25, the measured value by the CLEIA method (Control Example 3) was 669 pg / mL, while the measured value by the LTIA method (Comparative Example 6) with the addition of HeteroBlock was 816 pg / mL, and the measured value had a large deviation. The measured value by the LTIA method (Example 7) in which the anti-L chain λ monoclonal antibody of the present invention was added was 834 pg / mL, and the measured value did not change. The measured value by the LTIA method (Example 8) in which the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody were used in combination was 665 pg / mL, showing a tendency close to Control Example 3. The same result was obtained in sample number 26.

[0368] In sample number 27, the measured value by the CLEIA method (Control Example 3) was 410 pg / mL, while the measured value by the LTIA method (Comparative Example 6) with the addition of HeteroBlock was 1901 pg / mL, and the measured value had a large deviation. The measured value by the LTIA method (Example 7) in which the anti-L chain λ monoclonal antibody of the present invention was added was 1045 pg / mL, and the degree of deviation was reduced. The measured value by the LTIA method (Example 8) in which the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody were used in combination was 423 pg / mL, and the degree of deviation was further reduced, showing a value substantially the same as that of Control Example 3. The same tendency was obtained in samples 28 to 30.

[0369] (3) As described above, in the immunoassay method, non-specific reactions derived from the sample can be suppressed by allowing the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody of the present invention to be present in the immune reaction system. According to the present invention, non-specific reactions that cannot be suppressed by HeteroBlock, which is a commercially available non-specific reaction inhibitor, can be suppressed, so the non-specific reaction inhibitory effect of the anti-L chain λ monoclonal antibody or the anti-L chain κ monoclonal antibody of the present invention is an unexpected result. In addition, by using the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody in combination, an additive effect of suppressing non-specific reactions was confirmed.

[0370] [Experimental Example 4: Study on the concentration of non-specific reaction inhibitors in the LTIA method: Measurement of sIL-2R]

[0371] The sIL-2R concentration in the sample was measured using a reagent based on the LTIA method to which the anti-L chain λ monoclonal antibody and / or anti-L chain κ monoclonal antibody of the present invention was added at a low concentration, and the non-specific reaction inhibitory effect was confirmed.

[0372] The control samples (samples 1 to 6) and some deviation samples (samples 7, 8, and 10) used in Experimental Example 1 were used as samples. The effect was confirmed by comparing the results with those of Comparative Example 1 of Experimental Example 1.

[0373] [Example 9] (Measurement based on LTIA method: adding anti-L chain λ monoclonal antibody)

[0374] The measurement was performed in the same manner as in Comparative Example 1, except that the anti-L chain λ monoclonal antibody (S06215 antibody prepared in Reference Example 1) was added to the first reagent in Comparative Example 1 to a concentration of 20 μg / mL.

[0375] [Example 10] (Measurement based on LTIA method: adding anti-L chain λ monoclonal antibody)

[0376] The measurement was performed in the same manner as in Comparative Example 1, except that the anti-L chain λ monoclonal antibody (S06215 antibody prepared in Reference Example 1) was added to the first reagent in Comparative Example 1 at 50 μg / mL.

[0377] [Example 11] (Measurement based on LTIA method: adding anti-L chain κ monoclonal antibody)

[0378] The measurement was performed in the same manner as in Comparative Example 1, except that the anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) was added to the first reagent in Comparative Example 1 so as to have a concentration of 20 μg / mL.

[0379] [Example 12] (Measurement based on LTIA method: adding anti-L chain κ monoclonal antibody)

[0380] The measurement was performed in the same manner as in Comparative Example 1, except that the anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) was added to the first reagent in Comparative Example 1 so as to have a concentration of 50 μg / mL.

[0381] [Example 13] (Measurement based on LTIA method: adding anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody)

[0382] The measurement was performed in the same manner as in Comparative Example 1, except that the anti-L chain λ monoclonal antibody (S06215 antibody prepared in Reference Example 1) was added to the first reagent as in Comparative Example 1 so as to be 20 μg / mL, and the anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) was added to be 20 μg / mL. The measurement results are shown in Table 5.

[0383] [Example 14] (Measurement based on LTIA method: adding anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody)

[0384] The measurement was performed in the same manner as in Comparative Example 1, except that the anti-L chain λ monoclonal antibody (S06215 antibody prepared in Reference Example 1) was added to the first reagent as in Comparative Example 1 so as to be 50 μg / mL, and the anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) was added to be 50 μg / mL. The measurement results are shown in Table 5.

[0385] <Results and Investigation>

[0386] Based on the results of Comparative Example 1 and Examples 9 to 14, the effect of the non-specific reaction inhibitor of the present invention at a low concentration was examined.

[0387] (1) Measurement results for control samples (sample numbers 1 to 6)

[0388] All Examples are substantially the same as Comparative Example 1 which does not contain a non-specific reaction inhibitor. Here, according to the results of Experimental Example 1, Comparative Example 1 is the same as the measured value of the CLEIA method (Control Example 1), so it can be judged that all Examples are the same as Control Example 1. Therefore, it can be seen that the addition of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody of the present invention has no effect on the measured value of the control sample within the range of 20 to 50 μg / mL.

[0389] (2) Measurement results for deviation samples (sample numbers 7, 8, and 10)

[0390] The measured values ​​of the LTIA method without adding a non-specific reaction inhibitor in Sample No. 7, Sample No. 8, and Sample No. 10 (Comparative Example 1) were 1814 U / mL, 738 U / mL, and 1857 U / mL, respectively, which deviated from the measured values ​​of Comparative Example 1 as described above.

[0391] On the other hand, for sample number 7, to which the anti-L chain λ monoclonal antibody of the present invention was added at 20 μg / mL and 50 μg / mL, the measured values ​​by the LTIA method (Examples 9 and 10) were 1262 U / mL and 812 U / mL, respectively, which were lower values ​​than those in Comparative Example 1.

[0392] The measured values ​​of the LTIA method using a combination of 20 μg / mL of anti-L chain λ monoclonal antibody and 20 μg / mL of anti-L chain κ monoclonal antibody (Example 13) and the measured values ​​of the LTIA method using a combination of 50 μg / mL of anti-L chain λ monoclonal antibody and 50 μg / mL of anti-L chain κ monoclonal antibody (Example 14) were 1235 U / mL and 779 U / mL, respectively, which are lower values ​​than those in Comparative Example 1.

[0393] In addition, for sample number 10, to which the anti-L chain κ monoclonal antibody of the present invention was added at 20 μg / mL and 50 μg / mL, the measured values ​​by the LTIA method (Examples 11 and 12) were 1777 U / mL and 1674 U / mL, respectively, which were lower values ​​than those in Comparative Example 1.

[0394] The measured values ​​of the LTIA method using a combination of 20 μg / mL of anti-L chain λ monoclonal antibody and 20 μg / mL of anti-L chain κ monoclonal antibody (Example 13) and the measured values ​​of the LTIA method using a combination of 50 μg / mL of anti-L chain λ monoclonal antibody and 50 μg / mL of anti-L chain κ monoclonal antibody (Example 14) were 1796 U / mL and 1685 U / mL, respectively, which are lower values ​​than those in Comparative Example 1.

[0395] (3) As described above, it was found that in the immunoassay method, when the anti-L chain λ monoclonal antibody or anti-L chain κ monoclonal antibody of the present invention is present in the first reagent at a low concentration of 20 to 50 μg / mL and the weight of the anti-L chain λ monoclonal antibody or anti-L chain κ monoclonal antibody is 4.3 to 10.7 μg relative to 10 μL of the sample, non-specific reactions derived from the sample can be suppressed. In addition, when the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody are used in combination at the above concentrations, various non-specific reactions derived from the sample can be suppressed in the same manner.

[0396] [Table 5]

[0397] sIL-2R measurement results (U / mL)

[0398]

[0399] [Experimental Example 5: Study on the concentration of non-specific reaction inhibitors in the LTIA method: Measurement of sIL-2R]

[0400] The sIL-2R concentration in the sample was measured using a reagent based on the LTIA method to which the anti-L chain λ monoclonal antibody and / or anti-L chain κ monoclonal antibody of the present invention was added, and the non-specific reaction inhibitory effect was confirmed.

[0401] It should be noted that the following samples were used: samples that showed values ​​close to the measurement values ​​based on the chemiluminescent enzyme immunoassay (CLEIA method) (control example 1) (control samples: samples 31 to 33); and samples that showed non-specific reactions and had large deviations from the measurement values ​​of control example 1 (deviation samples: samples 34 to 37).

[0402] [Comparative Example 7] (Measurement based on the LTIA method: adding anti-human IgG, IgA, IgM polyclonal antibodies)

[0403] The measurement was performed in the same manner as in Comparative Example 1 except that commercially available anti-human IgG polyclonal antibody, anti-human IgA polyclonal antibody, and anti-human IgM polyclonal antibody (manufactured by SeraCare Life Sciences, Inc. S) were added to the first reagent in Comparative Example 1 to a concentration of 400 μg / mL. The measurement results are shown in Table 6.

[0404] [Example 15] (Measurement based on LTIA method: adding anti-L chain κ monoclonal antibody)

[0405] The measurement was performed in the same manner as in Comparative Example 1, except that the anti-L chain κ monoclonal antibody (S06215 antibody prepared in Reference Example 1) was added to the first reagent in Comparative Example 1 so as to have a concentration of 400 μg / mL. The measurement results are shown in Table 6.

[0406] [Example 12] (Measurement based on LTIA method: adding anti-L chain κ monoclonal antibody)

[0407] The measurement was performed in the same manner as in Comparative Example 1, except that the anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) was added to the first reagent in Comparative Example 1 so as to be 50 μg / mL. The measurement results are shown in Table 6. It should be noted that "ND" in Table 6 means No Data.

[0408] [Example 2] (Measurement based on LTIA method: adding anti-L chain κ monoclonal antibody)

[0409] The measurement was performed in the same manner as in Comparative Example 1, except that the anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) was added to the first reagent in Comparative Example 1 to a concentration of 200 μg / mL. The measurement results are shown in Table 6.

[0410] [Example 16] (Measurement based on LTIA method: adding anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody)

[0411] The measurement was performed in the same manner as in Comparative Example 1, except that the anti-L chain λ monoclonal antibody (S06215 antibody) and the anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) were added to the first reagent as in Comparative Example 1 at 400 μg / mL. The measurement results are shown in Table 6.

[0412] [Example 14] (Measurement based on LTIA method: adding anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody)

[0413] The measurement was performed in the same manner as in Comparative Example 1, except that the anti-L chain λ monoclonal antibody (S06215 antibody prepared in Reference Example 1) and the anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) were added to the first reagent as shown in Comparative Example 1 so as to be 50 μg / mL. The measurement results are shown in Table 6. It should be noted that "ND" in Table 6 means No Data.

[0414] [Example 3] (Measurement based on LTIA method: adding anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody)

[0415] The measurement was performed in the same manner as in Comparative Example 1, except that the anti-L chain λ monoclonal antibody (S06215 antibody prepared in Reference Example 1) and the anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) were added to the first reagent as in Comparative Example 1 so as to be 200 μg / mL. The measurement results are shown in Table 6.

[0416] [Table 6]

[0417] sIL-2R measurement results (U / mL)

[0418]

[0419] <Results and Investigation>

[0420] Based on the results of Control Example 1, Comparative Example 1, Comparative Example 7, Example 2, Example 3, and Examples 14 to 16, the effect of the non-specific reaction inhibitor of the present invention was examined.

[0421] (1) Measurement results for control samples (sample numbers 31 to 33)

[0422] All examples are substantially the same as Comparative Example 1 which does not contain a non-specific reaction inhibitor. Here, according to the results of Experimental Example 1, Comparative Example 1 is the same as the measured value of the CLEIA method (Control Example 1), so it can be judged that any example is the same as Control Example 1. In addition, from the results of Experimental Example 4, it can be seen that there is no effect on the measured value of the control sample within the range of 20 to 50 μg / mL. Therefore, it can be seen that with respect to the addition of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody of the present invention, there is no effect on the measured value of the control sample within the range of 20 to 400 μg / mL.

[0423] (2) Measurement results for deviation samples (sample numbers 34 to 37)

[0424] In sample numbers 34 to 37, the measured values ​​by the LTIA method (Comparative Example 1) without adding a non-specific reaction inhibitor deviated from the measured values ​​of Control Example 1 as described above.

[0425] In sample number 34, the measured value of the LTIA method to which 400 μg / mL of the anti-L chain κ monoclonal antibody of the present invention was added (Example 15) was 350 U / mL, and there was a tendency to be closer to Control Example 1 than Comparative Example 1. In addition, the measured value of the LTIA method using a combination of 50 μg / mL of anti-L chain λ monoclonal antibody and 50 μg / mL of anti-L chain κ monoclonal antibody (Example 14), the measured value of the LTIA method using a combination of 200 μg / mL of anti-L chain λ monoclonal antibody and 200 μg / mL of anti-L chain κ monoclonal antibody (Example 3), and the measured value of the LTIA method using a combination of 400 μg / mL of anti-L chain λ monoclonal antibody and 400 μg / mL of anti-L chain κ monoclonal antibody (Example 16) were 434 U / mL, 369 U / mL, and 410 U / mL, respectively, and there was a tendency to be closer to Control Example 1 than Comparative Example 1. The same tendency was confirmed in other samples.

[0426] In addition, in sample number 34, the measured values ​​of the LTIA method of the anti-L chain κ monoclonal antibody of the present invention added at 50 μg / mL, 200 μg / mL or 400 μg / mL (Example 12, Example 2, Example 15) were 419 U / mL, 372 U / mL, and 350 U / mL, respectively, showing a significant non-specific reaction inhibition effect at a lower added concentration than the anti-human IgG polyclonal antibody, anti-human IgA polyclonal antibody and anti-human IgM polyclonal antibody (Comparative Example 7). In addition, the measured values ​​of the LTIA method (Example 14) using anti-L chain λ monoclonal antibody 50 μg / mL and anti-L chain κ monoclonal antibody 50 μg / mL in combination, the measured values ​​of the LTIA method (Example 3) using anti-L chain λ monoclonal antibody 200 μg / mL and anti-L chain κ monoclonal antibody 200 μg / mL in combination, and the measured values ​​of the LTIA method (Example 16) using anti-L chain λ monoclonal antibody 400 μg / mL and anti-L chain κ monoclonal antibody 400 μg / mL in combination were 434 U / mL, 369 U / mL, and 410 U / mL, respectively, showing a significant non-specific reaction inhibitory effect at a lower added concentration than anti-human IgG polyclonal antibody, anti-human IgA polyclonal antibody, and anti-human IgM polyclonal antibody (Comparative Example 7). The same tendency was confirmed in other samples.

[0427] (3) As described above, it was found that in the immunoassay method, when the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody of the present invention are present in the first reagent at a concentration of 20 to 400 μg / mL and the weight of the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody is 4.3 to 85.7 μg relative to 10 μL of the sample, non-specific reactions derived from the sample can be suppressed. In addition, when the anti-L chain λ monoclonal antibody and the anti-L chain κ monoclonal antibody are used in combination at the above concentrations, various non-specific reactions derived from the sample can be suppressed in the same manner.

[0428] Furthermore, according to the present invention, non-specific reactions can be suppressed even at lower addition concentrations compared to anti-human IgG polyclonal antibodies, anti-human IgA polyclonal antibodies, and anti-human IgM polyclonal antibodies that are commercially available non-specific reaction inhibitors.

[0429] [Experimental Example 6: Study on the concentration of non-specific reaction inhibitors in ELISA method: Measurement of sIL-2R]

[0430] The non-specific reaction inhibitory effect was confirmed by measuring the sIL-2R concentration in the sample using a reagent based on the ELISA method to which the anti-L chain λ monoclonal antibody and / or anti-L chain κ monoclonal antibody of the present invention was added at a low concentration.

[0431] It should be noted that the following samples were used as samples: samples that showed values ​​close to the measurement values ​​based on the chemiluminescent enzyme immunoassay (CLEIA method) (control example 1) (control samples: samples 31 and 33); and samples that showed non-specific reactions and deviated from the measurement values ​​of control example 1 (deviation samples: samples 38 to 40).

[0432] <Method>

[0433] (1) Anti-sIL-2R monoclonal antibody (clone number 92212) 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. 100 μL of the dissolved solution was dispensed into each well of a 96-well microplate and allowed to stand at 4°C overnight.

[0434] (2) 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.

[0435] (3) After each well of the ELISA plate was washed three times with 400 μL of PBST, 100 μL of a sample diluted 40-fold with the following components added to BSA-PBST was added to each well and allowed to stand at room temperature for 1 hour.

[0436] (4) After washing each well three times with 400 μL of PBST, 100 μL of 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 allowed to stand at room temperature for 1 hour.

[0437] (5) After washing each well three times with 400 μL of PBST, 100 μL of HRP-labeled streptavidin (ThermoFisher Scientific) diluted to 0.20 μg / mL with BSA-PBST was dispensed into each well and allowed to stand at room temperature for 30 minutes.

[0438] (6) After washing each well three times with 400 μL of PBST, 50 μL of a citrate buffer solution (pH 5.0) containing 0.2% o-phenylenediamine and 0.02% hydrogen peroxide was added. After standing at room temperature for 10 minutes, 50 μL of 4.5 N sulfuric acid was added to stop the enzyme reaction, and the absorbance at a wavelength of 492 nm was measured.

[0439] (7) Using a standard substance with a known concentration as a calibrator, calculate the sIL-2R value in the sample being tested.

[0440] (8) Using the measurement results of the calibrator, a calibration curve for each test using each reagent was prepared. Using the obtained calibration curve, the measurement value of each sample was obtained. For each sample, the value obtained by dividing the measurement value in Comparative Example 9, Comparative Example 10, and Example 17 by the measurement value of Control Example 1 was calculated and evaluated as a relative ratio (%). When the relative ratio was 90% or more and 110% or less, it was determined that there was no deviation relative to the measurement value of Control Example 1.

[0441] [Comparative Example 9] (Measurement by ELISA method: without additives)

[0442] The measurement was performed using BSA-PBST as a sample diluent. The results of the measurement values ​​are shown in Table 7, and the results of the comparison are shown in Table 8.

[0443] [Comparative Example 10] (Measurement by ELISA method: Addition of HBR-1)

[0444] The measurement was performed in the same manner as in Comparative Example 9 except that HBR-1 (manufactured by SCANTIBODIES LABORATORY, INC.), a commercially available nonspecific reaction inhibitor, was added to the sample dilution in Comparative Example 9 to give a concentration of 400 μg / mL. The measurement results are shown in Table 7, and the comparative results are shown in Table 8.

[0445] [Example 17] (Measurement by ELISA method: adding anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody)

[0446] The measurement was performed by the same method as in Comparative Example 9, except that the anti-L chain λ monoclonal antibody (S06215 antibody prepared in Reference Example 1) and the anti-L chain κ monoclonal antibody (S06205 antibody prepared in Reference Example 1) were added to the sample dilution solution shown in Comparative Example 9 so as to be 200 μg / mL. The results of the measured values ​​are shown in Table 7, and the results of the relative comparison with Example 1 are shown in Table 8.

[0447] [Table 7]

[0448] sIL-2R measurement results (U / mL)

[0449]

[0450] [Table 8]

[0451] sIL-2R assay results (%)

[0452]

[0453] <Results and Investigation>

[0454] The effect of the non-specific reaction inhibitor of the present invention was examined based on the results of Control Example 1, Comparative Example 9, Comparative Example 10 and Example 17. The ratio (relative ratio) of the measured value of the ELISA method without adding additives (Comparative Example 9) to the measured value of the CLEIA method (Control Example 1) of the control samples 31 and 33 was 99 to 104%, and the control samples 31 and 33 were evaluated as control samples that did not produce non-specific reactions in the ELISA method. In addition, the ratio (relative ratio) of the measured value of the ELISA method without adding additives (Comparative Example 9) to the measured value of the CLEIA method (Control Example 1) of the deviation samples 38 to 40 was 110 to 115%, and the deviation samples 38 to 40 were evaluated as deviation samples that produced non-specific reactions in the ELISA method.

[0455] (1) Measurement results for control samples (sample numbers 31 and 33)

[0456] All Examples were substantially the same as Control Example 1 and Comparative Example 9 which did not contain a non-specific reaction inhibitor. Therefore, it was found that the addition of the anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody of the present invention (200 μg / mL each) had no effect on the measured values ​​of the control sample.

[0457] (2) Measurement results for deviation samples (sample numbers 38 to 40)

[0458] In sample numbers 38 to 40, it was found that the measured values ​​of the ELISA method without adding a non-specific reaction inhibitor (Comparative Example 9) deviated from the measured values ​​of Control Example 1.

[0459] Regarding sample number 38, the measurement value of the ELISA method (Comparative Example 10) in which 400 μg / mL of HBR-1 was added was 1088 U / mL, indicating that the non-specific inhibitory effect was insufficient.

[0460] The measured value of the ELISA method (Example 17) using a combination of 200 μg / mL of the anti-L chain λ monoclonal antibody of the present invention and 200 μg / mL of the anti-L chain κ monoclonal antibody was 884 U / mL, which is a lower value than that of Comparative Example 9. The same tendency was obtained for other deviation samples. When the component of the present invention was added to the sample diluent in the ELISA reagent as a measurement method with B / F separation, the consistency of the measured value of the CLEIA method as a measurement method with B / F separation was improved.

[0461] According to the present invention, nonspecific reactions that cannot be inhibited by HBR-1, a commercially available nonspecific reaction inhibitor, can be inhibited. Therefore, the nonspecific reaction inhibitory effect of the anti-L chain λ monoclonal antibody and anti-L chain κ monoclonal antibody of the present invention provides results exceeding expectations.

[0462] (3) As described above, when the component of the present invention is added to a sample diluent in an ELISA reagent as a measurement method involving B / F separation, the consistency of the measurement value with respect to the CLEIA method is improved.

[0463] Generally, non-specific reactions are less likely to occur when B / F separation is performed, but even slightly occurring non-specific reactions may be suppressed by this component.

[0464] Industrial Applicability

[0465] According to the present invention, by carrying out an immune reaction in the presence of both or either of an anti-L chain λ monoclonal antibody and an anti-L chain κ monoclonal antibody, even when a sample contains nonspecific factors, nonspecific reactions that cannot be eliminated by conventional nonspecific reaction inhibitors can be suppressed, thereby achieving an accurate immunoassay method.

Claims

1. An immunological assay method, wherein: In the method of immunologically measuring a substance to be measured in a sample, an immune reaction is carried out in the presence of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody, in the presence of an anti-immunoglobulin L chain λ monoclonal antibody, or in the presence of an anti-immunoglobulin L chain κ monoclonal antibody. However, the substance to be measured does not include anti-immunoglobulin L chain λ antibody and anti-immunoglobulin L chain κ antibody. 2 . The immunological assay method according to claim 1 , which is an immunological assay method based on a homogeneous method or an immunological assay method based on a heterogeneous method.

3. The immunological assay method according to claim 2, comprising: a step of contacting a sample containing a substance to be measured with the anti-immunoglobulin L chain λ monoclonal antibody or the anti-immunoglobulin L chain κ monoclonal antibody; a step of adding a specific binding partner for the substance to be measured; and A step of detecting a signal derived from a reaction between the analyte in the solution and the specific binding partner.

4. The immunological assay method according to claim 2, wherein: The immunological determination method based on the homogeneous method is latex immunoturbidimetry.

5. The immunological assay method according to claim 4, comprising: a step of contacting a sample containing a substance to be measured with the anti-immunoglobulin L chain λ monoclonal antibody or the anti-immunoglobulin L chain κ monoclonal antibody in a solution; a step of adding latex particles carrying a specific binding partner for the substance to be measured to the solution; and A step of optically detecting the degree of aggregation of the latex particles in the solution.

6. A method for suppressing nonspecific reactions in a method for immunologically measuring a substance to be measured in a sample, wherein: The immune reaction is carried out in the presence of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody, in the presence of an anti-immunoglobulin L chain λ monoclonal antibody, or in the presence of an anti-immunoglobulin L chain κ monoclonal antibody.

7. The method for inhibiting nonspecific reactions according to claim 6, wherein: The method for performing immunological assay is an immunological assay method based on a homogeneous method or an immunological assay method based on a heterogeneous method.

8. The method for inhibiting nonspecific reactions according to claim 7, wherein: The immunological determination method based on the homogeneous method is latex immunoturbidimetry.

9. The method for inhibiting nonspecific reactions according to claim 8, comprising: a step of contacting a sample containing a substance to be measured with the anti-immunoglobulin L chain λ monoclonal antibody or the anti-immunoglobulin L chain κ monoclonal antibody in a solution; a step of adding latex particles carrying a specific binding partner for the substance to be measured to the solution; and A step of optically detecting the degree of aggregation of the latex particles in the solution.

10. An immunological assay reagent comprising either or both of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody. The immunological assay reagent according to claim 10, which is used for an immunological assay method based on a homogeneous method or an immunological assay method based on a heterogeneous method.

12. The immunoassay reagent according to claim 11, wherein The immunological determination method based on the homogeneous method is latex immunoturbidimetry.

13. An immunological assay kit comprising either or both of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody. The immunological assay kit according to claim 13, which is used for an immunological assay method based on a homogeneous method or an immunological assay method based on a heterogeneous method.

15. The immunoassay kit according to claim 14, wherein The immunological determination method based on the homogeneous method is latex immunoturbidimetry.

16. The immunoassay kit according to claim 14, wherein The homogeneous method is latex immunoturbidimetry. The immunological assay kit comprises the following reagents: (1) a first reagent comprising either or both of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody; (2) A second reagent comprising latex particles supporting a specific binding partner for a substance to be measured.

17. A composition comprising either or both of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody.

18. A non-specific reaction inhibitor for an immunological assay method, comprising as an active ingredient either or both of an anti-immunoglobulin L chain λ monoclonal antibody and an anti-immunoglobulin L chain κ monoclonal antibody.

19. Use of the composition according to claim 17 as a non-specific reaction inhibitor in an immunological assay method.

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