Immunological assay methods, immunoassay kits, and non-specific reaction inhibitors

Abstract

To provide an immunological measurement method, an immunological measurement kit, and a nonspecific reaction inhibitor for immunological measurement that have an effect of suppressing nonspecific reactions. [Solution] An immunoassay method for measuring a target substance contained in a sample, A step of bringing the aforementioned sample into contact with the anti-C4BP antibody. A step of bringing a detection substance that specifically binds to the substance to be measured into contact with the sample, and The process includes measuring a signal corresponding to the content or titer of the substance to be measured, The immunological measurement method described above.

Description

Technical Field

[0001] The present invention relates to an immunological measurement method, an immunological measurement kit, and a non-specific reaction inhibitor.

Background Art

[0002] Conventionally, biochemical measurement methods are known as means for detecting various components contained in human biological samples. Among biochemical measurement methods, immunological measurement methods are highly specific measurement methods because they utilize specific binding between proteins such as antigen-antibody reactions.

[0003] Since various substances are present in a sample, binding that is not based on the original specific reaction may occur, or the specific immune reaction may be hindered. Such a reaction that is not the original specific reaction is called a non-specific reaction, and the causative substance may be called a non-specific factor. When a non-specific reaction occurs in biochemical measurement, a measurement error occurs. Therefore, various non-specific factors have been elucidated so far, and methods for suppressing non-specific reactions have been studied.

[0004] For example, heterophilic antibodies are known as non-specific factors (Non-Patent Document 1). Heterophilic antibodies are a general term for human antibodies that show reactivity with animal-derived antibodies, which are the main components of immunological measurement methods, and HAMA (human anti-mouse immunoglobulin antibody) is known as a representative one. In addition, rheumatoid factor (RF) is also known as a non-specific factor. Rheumatoid factor is mainly an IgM-class antibody that recognizes the Fc region of IgG, and it is present not only in the blood of patients with rheumatoid arthritis but also in the blood of healthy individuals. As a method for suppressing non-specific reactions caused by heterophilic antibodies or rheumatoid factors, a method of pre-mixing an anti-human IgM antibody, an anti-human IgA antibody, or an anti-human IgG antibody with a sample is known (Patent Document 1).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] [Non-Patent Document 1] Biophysical Chemistry, 2007, Vol. 51, pp. 223-226 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The method of capturing IgM and other substances using anti-human IgM antibodies, as described in Patent Document 1, and suppressing nonspecific reactions is currently a method that has been put into practical use with various reagents. However, there are still nonspecific reactions that cannot be suppressed even by these antibodies.

[0008] The object of the present invention is to provide an immunological measurement method, an immunological measurement kit, and an immunological nonspecific reaction inhibitor that have a nonspecific reaction inhibitory effect. [Means for solving the problem]

[0009] The present invention has the following aspects. [1] An immunoassay method for measuring a target substance contained in a sample, A step of bringing the aforementioned sample into contact with the anti-C4BP antibody. A step of bringing a detection substance that specifically binds to the substance to be measured into contact with the sample, and The process includes measuring a signal corresponding to the content or titer of the substance to be measured, The immunological measurement method described above. [2] The sample contains C4BP at a concentration of 20 μg / mL or higher. The immunological measurement method described in [1]. [3] The anti-C4BP antibody is a polyclonal antibody. The immunological measurement method described in [1] or [2]. [4] The anti-C4BP antibody is an anti-C4BPα antibody. An immunoassay method described in any one of [1] to [3]. [5] The step of bringing the sample into contact with the anti-C4BP antibody is This includes adjusting the concentration of free C4BP, which is not bound to the anti-C4BP antibody, in the aforementioned sample to 20 μg / mL or less. An immunoassay method described in any one of [1] to [4]. [6] The step of bringing the sample into contact with the anti-C4BP antibody, The step of bringing the detection substance, which specifically binds to the substance to be measured, into contact with the sample, Perform these actions simultaneously or in this order. An immunoassay method described in any one of [1] to [5]. [7] The immunoassay method is homogeneous. An immunoassay method described in any one of [1] to [6]. [8] Anti-C4BP antibody, A detection substance that specifically binds to the substance to be measured, Immunological measurement kit. [9] The first reagent containing the anti-C4BP antibody, A second reagent containing the aforementioned detection substance, The immunoassay kit described in [8].

[10] The first reagent contains the anti-C4BP antibody, It is contained at a concentration of 1 to 200 μg / mL. [9] Immunological assay kit as described.

[11] Nonspecific reaction inhibitors for immunoassays, including anti-C4BP antibodies. [Effects of the Invention]

[0010] According to the present invention, an immunological measurement method, an immunological measurement kit, and an immunological nonspecific reaction inhibitor can be provided, all of which have a nonspecific reaction inhibitory effect. [Modes for carrying out the invention]

[0011] Hereinafter, embodiments of the present invention will be specifically described. However, the present invention is not limited to the following embodiments, and can be variously modified and implemented within the scope of the gist.

[0012] Hereinafter, in this specification, "react" and "bind" are used in the same meaning.

[0013] When a numerical range is described as, for example, "1-10" or "1~10", it means a range from 1 to 10, and means a numerical range including the lower limit value 1 and the upper limit value 10. Also, the upper limit value and the lower limit value of the numerical range can be arbitrarily combined. Further, the numerical ranges with respect to each physical property, composition, measurement process, etc. can be arbitrarily combined.

[0014] <Immunological measurement method> The immunological measurement method according to the first aspect of the present invention is a method for measuring a substance to be measured contained in a sample, including a step of contacting the sample with an anti-C4BP antibody, a step of contacting a detection substance that specifically binds to the substance to be measured with the sample, and a step of measuring a signal corresponding to the content or titer of the substance to be measured.

[0015] [Sample] In the present invention, the sample containing the substance to be measured is preferably a biological sample. The biological sample is not particularly limited as long as it is a sample derived from a living body, and a liquid sample such as a body fluid is preferred. At least one selected from the group consisting of blood, plasma, serum, urine, cerebrospinal fluid, saliva, sweat, ascites, nasal discharge, feces, and an extract of cells or tissues is more preferred, and any one selected from the group consisting of blood, plasma, and serum is even more preferred. The living body is preferably a mammal such as a human, mouse, guinea pig, rat, monkey, dog, cat, hamster, horse, cow, and pig, and more preferably a human. The biological sample may be collected or prepared at the time of implementing the present invention, or may be collected or prepared in advance and stored. The biological sample may be diluted with a liquid such as water, a buffer solution, and physiological saline.

[0016] [Substances to be measured] In this embodiment, there are no particular limitations on the substances to be measured contained in the sample, and examples include proteins (antigens, haptens, antibodies), carbohydrates, lipids, nucleic acids, and chemical substances (hormones, drugs). Among these, antigens or antibodies are preferred, and antigens consisting of proteins are more preferred. Examples of antigens consisting of proteins include soluble interleukin-2 receptor (sIL-2R), CRP, fibrin and fibrinogen degradation products, D-dimer, soluble fibrin (SF), lipoprotein(a) (Lp(a)), matrix metalloproteinase-3 (MMP-3), prostate-specific antigen (PSA), IgG, IgA, IgM, IgE, IgD, anti-streptolysin O antibody, rheumatoid factor, transferrin, haptoglobin, α1-antitrypsin, α1-acid glycoprotein, α2- Examples include chloroglobulin, hemopexin, antithrombin-III, alpha-fetoprotein, CEA (calcinoembryonic antigen), ferritin, HBs-Ag (hepatitis B outer antigen), Anti-HBs (anti-hepatitis B outer antibody), HBe-Ag (hepatitis B e antigen), Anti-HBe (anti-hepatitis B e antibody), Anti-HBc (anti-hepatitis B core antibody), SARS-CoV-2, human brain natriuretic peptide (BNP), pulmonary surfactant protein D (SP-D), and procalcitonin (PCT).

[0017] [Non-specific reactants] In the present invention, a nonspecific reactant is a substance that reacts or adsorbs nonspecifically to a substance involved in immunological measurement or to an instrument used in immunological measurement. Examples of substances involved in immunological measurement include the substance to be measured, a detection substance that specifically binds to the substance to be measured, and a capture substance. Examples of instruments used in immunological measurement include a reaction vessel and a stirring device.

[0018] The inventors of this invention conducted thorough research and found that samples containing a large amount of C4BP were found to reduce the accuracy of the measurement of the target substance in immunological measurements. Therefore, C4BP was considered to be a non-specific reactant. C4BP is a protein commonly known as a complement regulatory factor present in human serum, and is also called C4b-binding protein. It can form a complex with a molecular weight of 570 kDa in serum, and may contain a 75 kDa α chain as well as a 40 kDa β chain. In this embodiment, since an anti-C4BP antibody that specifically binds to C4BP is used, nonspecific reactions can be effectively suppressed when the sample contains C4BP, which is a nonspecific reactant.

[0019] When the C4BP concentration in the sample is 20 μg / mL or higher, the likelihood of a nonspecific reaction increases, and when it is 40 μg / mL or higher, the likelihood of a nonspecific reaction increases even further. In conventional methods, when the C4BP concentration in the sample is above the lower limit mentioned above, nonspecific reactions cannot be suppressed, and the measurement accuracy of the target substance may decrease. On the other hand, in this embodiment, since an anti-C4BP antibody is used, nonspecific reactions can be suppressed even when the C4BP concentration in the sample is above the lower limit mentioned above, and the measurement accuracy of the target substance is improved.

[0020] [Anti-C4BP antibody] The anti-C4BP antibody in this embodiment may be an antibody that binds to a 570 kDa C4BP complex, an antibody that binds to the α-chain of C4BP, or an antibody that binds to the β-chain.

[0021] In this embodiment, the anti-C4BP antibody may be a polyclonal antibody or a monoclonal antibody, but it is preferably a polyclonal antibody. When the anti-C4BP antibody in this embodiment is a polyclonal antibody, multiple antibodies can bind to multiple epitopes of C4BP simultaneously, which is advantageous in that it suppresses nonspecific reactions at a high rate and can react with various forms of C4BP.

[0022] In this embodiment, there are no particular limitations on the subclass of the anti-C4BP antibody, and examples include anti-C4BPα antibody and anti-C4BPβ antibody, with anti-C4BPα antibody being preferred. Since C4BP is composed of seven α chains and one β chain, if the anti-C4BP antibody in this embodiment is an anti-C4BPα antibody, the probability of the anti-C4BP antibody capturing C4BP increases.

[0023] In this embodiment, methods for bringing a sample into contact with an anti-C4BP antibody include using the anti-C4BP antibody as one of the reagent components of an immunoassay reagent, and adding the anti-C4BP antibody to an immunoassay reagent such as a sample diluent, a sample pretreatment solution, or a detection reagent containing a detection substance.

[0024] If the immunological assay method is, for example, latex immunoturbidimetric assay (LTIA), the sample may be mixed with the immunological assay reagent containing anti-C4BP antibody, or the sample may be mixed with a sample pretreatment solution containing anti-C4BP antibody beforehand, and then mixed with the immunological assay reagent.

[0025] If the immunoassay method is, for example, an enzyme-linked immunosorbent assay (ELISA), the sample may be mixed with a sample pretreatment solution containing anti-C4BP antibody before being dropped onto the microplate, or the sample may be mixed with a solution containing anti-C4BP antibody and detection antibody before being dropped onto the microplate.

[0026] If the immunological assay method is, for example, chemiluminescence, the sample may be mixed with a sample pretreatment solution containing anti-C4BP antibody beforehand, and then mixed with the immunoassay reagent. Alternatively, the immunoassay reagent (for example, a solution containing detection antibody or antigen, magnetic particles, etc.) may contain anti-C4BP antibody.

[0027] If the immunoassay method is performed on a solid phase, such as immunochromatography, the sample may be mixed with a sample pretreatment solution containing anti-C4BP antibody beforehand and then dropped onto the immunochromatographic test piece. Alternatively, the anti-C4BP antibody may be kept dry on the constituent material of the immunochromatographic test piece, such as a sample pad, and the sample may be mixed with the dissolved anti-C4BP antibody by dropping the sample onto the test piece.

[0028] In this embodiment, the amount of anti-C4BP antibody that comes into contact with the sample is not particularly limited, as long as it does not strongly affect the immune reaction between the substance to be measured and the detection substance, and can exert the desired non-specific reaction suppression effect. It can be set appropriately depending on the type of substance to be measured and the sample. In this embodiment, the amount of anti-C4BP antibody that comes into contact with the sample varies depending on the reagent composition, but it is preferable that the weight of anti-C4BP antibody per 10 μL of sample be 0.1 to 50 μg, more preferably 1 to 30 μg, and even more preferably 2 to 20 μg.

[0029] In this embodiment, one type of anti-C4BP antibody may be used alone, or two or more types may be used in combination, a monoclonal antibody and a polyclonal antibody may be used in combination, or an anti-C4BP antibody conjugated to an insoluble carrier may be used. When two or more types of anti-C4BP antibodies are used in combination, the total concentration of the multiple types of anti-C4BP antibodies should be within the preferred range described above.

[0030] In this embodiment, when the nonspecific reactive substance contained in the sample is C4BP, it is preferable to adjust the concentration of free C4BP contained in the sample to 40 μg / mL or less, more preferably to 20 μg / mL or less, and even more preferably to 10 μg / mL or less, by contacting the C4BP contained in the sample with the anti-C4BP antibody in a step of contacting the sample with the anti-C4BP antibody. Here, "free C4BP" refers to C4BP that is not bound to the anti-C4BP antibody and is detectable by the C4BP measurement reagent. By adjusting the concentration of free C4BP contained in the sample to be below the above upper limit, nonspecific reactions between the target substance or detection substance, etc., and C4BP are further suppressed, and the measurement accuracy of the target substance is further improved.

[0031] [Survey material for detection] In this invention, the detection substance is a substance other than an anti-C4BP antibody that specifically binds to the substance to be measured. The detection substance is used to make the substance to be measured measurable by the immunological measurement method described later.

[0032] Examples of detection substances include proteins other than anti-C4BP antibodies, peptides, amino acids, lipids, carbohydrates, nucleic acids, haptens, etc. The detection substance is preferably a protein, and more preferably an antibody.

[0033] The antibody used as the detection substance may be either a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred. Antibodies used as detection substances may be those obtained through an immunization process on common animals (mice, goats, sheep, etc.), or antibodies (chimeric antibodies, humanized antibodies, or fully humanized antibodies, etc.) in which the amino acid sequence of the immunogen (substance to be measured) has been altered to that of an animal species different from the animal immunized by the immunogen using genetic engineering technology. Antibodies used as detection materials can include not only the entire antibody molecule but also functional fragments of antibodies that possess antigen-antibody reaction activity. Examples of functional antibody fragments that possess antigen-antibody reaction activity include F(ab')2, Fab', single-chain antibodies (scFv), VHH (variable domain of heavy chain of heavy chain antibody) antibodies, and IgNAR (new antigen receptor) antibodies. These functional antibody fragments can be produced by treating antibodies obtained as described above with proteolytic enzymes (e.g., pepsin or papain).

[0034] The detection substance may be used alone or in combination of two or more types. The type and amount of detection substance used can be appropriately selected depending on the immunoassay method employed.

[0035] In this embodiment, the step of contacting the sample with the anti-C4BP antibody may be followed by the step of contacting the sample with a detection substance that specifically binds to the substance to be measured. Alternatively, the step of contacting the sample with the anti-C4BP antibody and the step of contacting the sample with a detection substance that specifically binds to the substance to be measured may be performed simultaneously. Alternatively, the step of contacting the sample with the anti-C4BP antibody may be performed after the step of contacting the sample with a detection substance that specifically binds to the substance to be measured. It is preferable that the step of contacting the sample with the anti-C4BP antibody and the step of contacting the sample with a detection substance that specifically binds to the substance to be measured be performed simultaneously or in this order, and more preferably in this order.

[0036] The solution obtained by the steps of contacting the sample with the anti-C4BP antibody and contacting the sample with a detection substance that specifically binds to the substance to be measured is sometimes referred to as the sample solution. The concentration of the anti-C4BP antibody in the sample solution is preferably 1 to 50 μg / mL, and more preferably 5 to 20 μg / mL.

[0037] [Immunological measurement method] In the step of measuring the signal corresponding to the content or titer of the target substance contained in the sample solution, the signal corresponding to the content or titer of the target substance contained in the sample solution is measured by an immunological assay method. Here, titer is an indicator that shows the concentration or activity of a particular substance.

[0038] There are no particular limitations on the type of immunological assay method, and various known methods can be appropriately selected. Examples of immunological assay methods include latex immunoturbidimetric assay (LTIA), immunoturbidimetric assay (TIA), electrochemiluminescence assay (ECLIA), chemiluminescence assay (CLIA), enzyme-linked immunosorbent assay (ELISA), and immunochromatography.

[0039] Generally, immunological measurements are broadly classified into homogeneous and heterogeneous methods. Homogeneous methods do not involve a washing process (hereinafter sometimes referred to as "(B) / (F) separation process") to separate the complex formed by the specific binding of the substance to be measured and the detection substance from the complex formed by the nonspecific binding of the substance to be measured and nonspecific reactants. Therefore, homogeneous methods are susceptible to the influence of nonspecific binding. Heterogeneous methods, because they perform the (B) / (F) separation process, are less susceptible to the influence of nonspecific binding.

[0040] The LTIA method is a type of homogeneous method. Methods for measuring target substances using the LTIA method can be broadly classified into two categories. The first method involves reacting latex particles immobilized with a detection substance for the substance to be measured with the substance to be measured to form a sandwich-type immune complex, and measuring the substance to be measured based on the degree of aggregation of the latex particles accompanying the formation of the immune complex. The second method involves adding proteins or other substances immobilized with multiple target substances or their analogues (including fragments thereof) to a reagent, and creating competition between these and the target substances in the sample. This inhibits the formation of an immune complex between the target substances in the reagent and latex particles immobilized with a detection substance that binds to the target substances, and the target substances are measured based on the degree of inhibition of latex particle aggregation due to the inhibition of immune complex formation. This embodiment can be used in any of the above methods.

[0041] In the LTIA method, specifically the method for measuring the target substance based on the degree of aggregation of latex particles associated with immune complex formation, the steps are as follows: mixing the sample with a solution containing anti-C4BP antibody; obtaining a sample solution by adding latex particles carrying the detection substance to the mixture; and optically detecting the degree of aggregation of latex particles in the sample solution.

[0042] In the LTIA method, the target substance can be measured by optically or electrochemically observing the degree of aggregation that occurs. Optical observation methods include measuring scattered light intensity, absorbance, or transmitted light intensity with optical instruments (endpoint method, rate method, etc.). The absorbance measurement obtained by measuring the sample solution is compared with the absorbance measurement obtained by measuring a standard substance (a sample in which the concentration of the target substance is known) to calculate the concentration (quantitative value) of the target substance contained in the sample. The measurement of absorbance, etc., of transmitted or scattered light may be performed using one wavelength or two wavelengths (difference or ratio between two wavelengths). The measurement wavelength is generally selected from 500 nm to 900 nm.

[0043] The measurement of the target substance in the sample solution may be performed manually or using a measuring device. The measuring device may be a general-purpose automated analyzer or a dedicated measuring device (dedicated instrument). Furthermore, it is preferable to carry out this measurement using a method that involves multiple operating steps, such as a two-step method (two-reagent method).

[0044] When the immunoassay method in this embodiment is the LTIA method, the detection substance can be immobilized and supported on latex particles by known methods such as physical adsorption, chemical bonding, or a combination thereof.

[0045] In the case of the physical adsorption method, the detection can be carried out by mixing the detection substance for the substance to be measured with latex particles in a solution such as a buffer solution and bringing them into contact, or by bringing the detection substance for the substance to be measured, dissolved in a buffer solution, into contact with a carrier, according to known methods.

[0046] Furthermore, when performing the test using a chemical binding method, it can be carried out by following known methods described in publications such as "Special Issue No. 53 of Clinical Pathology: Immunoassays for Clinical Tests - Techniques and Applications," edited by the Japanese Society of Clinical Pathology, published by the Clinical Pathology Publication Association in 1983; and "New Biochemistry Experiment Course 1: Protein IV," edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin in 1991. This involves mixing and contacting a specific binding partner for the target substance and a carrier with a divalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide, and then reacting the amino group, carboxyl group, thiol group, aldehyde group, or hydroxyl group of the specific binding partner for the target substance and the carrier with the aforementioned divalent crosslinking reagent.

[0047] There are no particular limitations on the synthetic polymers that constitute the latex particles, but examples include polystyrene, styrene-styrene sulfonate copolymers, methacrylic acid polymers, acrylic acid polymers, itaconic acid polymers, and styrene-hydrophilic carboxymonomer copolymers: for example, styrene-methacrylic acid copolymers, styrene-acrylic acid copolymers, and styrene-itaconic acid copolymers. Among these, styrene-methacrylic acid copolymers, styrene-itaconic acid copolymers, and styrene and styrene-styrene sulfonate copolymers are preferred, and styrene and styrene-(meth)acrylic acid copolymers are more preferred.

[0048] It is preferable that the detection substances for the target substance supported by the latex particles consist of multiple types in order to form a sandwich. If the target substance has multiple antibody recognition sites, one specific binding partner may suffice. For example, if the specific binding partner is a monoclonal antibody, multiple monoclonal antibodies with different recognition sites may be used. Also, for example, if the specific binding partner is a polyclonal antibody, it may be a polyclonal antibody derived from one type of antiserum, or from multiple types of antiserum. Furthermore, a combination of monoclonal antibodies and polyclonal antibodies may be used.

[0049] Furthermore, if it is necessary to perform treatment to suppress spontaneous aggregation of latex particles or nonspecific reactions, the latex particles may be treated by known methods, such as contacting and coating the surface with proteins such as bovine serum albumin (BSA), casein, gelatin, ovalbumin or its salts, surfactants, or skim milk powder, to perform a blocking treatment (masking treatment) of the carrier.

[0050] The TIA method is a type of homogeneous detection method in which, for example, antibodies are used as detection substances, and the aggregation of antibodies in the sample solution, depending on the amount of the target substance contained in the sample solution, increases the turbidity of the sample solution, which is detected as an optical signal.

[0051] Immunochromatography is a type of homogeneous method. For example, it uses gold colloid particles bound to a first antibody (a detection substance) and a membrane on which a second antibody (also a detection substance) is immobilized. A complex of gold colloid-labeled antibody, the substance to be measured, and the immobilized antibody is formed on the membrane, and the signal originating from the gold colloid particles is detected.

[0052] The ECLIA method is a type of heterogeneous method. For example, it uses magnetic particles bound to a first antibody (a detection substance) and a second antibody (a detection substance) bound to a metal complex such as ruthenium. A complex of magnetic particles, the substance to be measured, and the metal complex is formed in a sample solution, and then (B) / (F) separation is performed using the magnetism of the magnetic particles. Subsequently, the electrochemical emission amount, which increases in proportion to the amount of the complex (amount of metal complex), is detected as an optical signal.

[0053] The CLIA method is a type of heterogeneous method. For example, it uses magnetic particles conjugated with a first antibody (a detection substance) and a second antibody labeled with a chemiluminescent compound (also a detection substance). A complex of magnetic particles, the substance to be measured, and the chemiluminescent compound is formed in a sample solution, and then (B) / (F) separation is performed using the magnetism of the magnetic particles. Subsequently, the amount of chemical emission, which increases in proportion to the amount of the complex (amount of chemiluminescent compound), is detected as an optical signal.

[0054] ELISA is a type of heterogeneous assay that involves capturing antigens or antibodies, which are the target substances in a sample, using a detection substance, and then detecting the signal using an enzymatic reaction. Examples of ELISA methods include the direct method, indirect method, sandwich method, and competitive method.

[0055] In the sandwich method of ELISA, for example, the steps of contacting the target substance with an antibody that specifically binds to the target substance, contacting the target substance with an antibody labeled with a labeling substance (detection substance), and measuring the target substance by detecting a signal corresponding to the labeling substance.

[0056] If the immunoassay method in this embodiment is the ELISA method, the anti-C4BP antibody may be added, for example, to the sample diluent or pretreatment solution, or to the solution in which the antigen-antibody reaction is performed. Specific methods, such as the method for immobilizing the antibody onto an insoluble carrier and the method for binding the antibody to the labeling substance, can be those well known to those skilled in the art.

[0057] The immunological assay method of this embodiment can suppress nonspecific reactions between the target substance and nonspecific reactants without performing the (B) / (F) separation process, thereby improving the accuracy of the measurement of the target substance. Therefore, the immunological assay method of this embodiment is preferably a homogeneous method, more preferably an LTIA method or an immunochromatography method, and most preferably an LTIA method.

[0058] <Immunological assay kit> An immunological measurement kit according to a second aspect of the present invention comprises the aforementioned anti-C4BP antibody and a detection substance that specifically binds to the substance to be measured. In this aspect, the anti-C4BP antibody and the detection substance may be contained in a single reagent or in separate reagents. The immunological measurement kit preferably comprises a first reagent containing the anti-C4BP antibody and a second reagent containing the detection substance. The first and second reagents may be solutions, freeze-dried solutions, or solutions that have been dried and stored on plates, pads, etc.

[0059] The amount of anti-C4BP antibody contained in the immunological assay kit is not particularly limited, as long as it does not strongly affect the immune reaction between the substance to be measured and the detection substance, and can exert the desired non-specific reaction suppression effect. It can be appropriately set depending on the type of substance to be measured and the sample. The concentration of anti-C4BP antibody in the sample solution obtained using the immunological assay kit is preferably 1 to 50 μg / mL, and more preferably 5 to 20 μg / mL.

[0060] The immunological measurement kit according to this embodiment may include components other than the anti-C4BP antibody and the detection substance in its kit composition. Examples of components other than the anti-C4BP antibody and the detection substance include buffers, proteins, peptides, amino acids, nucleic acids, lipids, phospholipids, carbohydrates, inorganic salts, polymer compounds, surfactants, other nonspecific reaction inhibitors, preservatives, etc.

[0061] There are no particular limitations on the type of buffer used; any buffer commonly used in the field of biochemical measurements can be appropriately selected. Examples of buffers include imidazole buffer, phosphate buffer (PBS), Tris buffer, Bis-Tris buffer, MOPS buffer, MES buffer, ADA buffer, PIPES buffer, ACES buffer, BES buffer, TES buffer, HEPES buffer, acetamidoglycine buffer, Tricinamide buffer, glycinamide buffer, and Bicine buffer. These buffers may be used individually or in combination of two or more types.

[0062] The hydrogen ion concentration of the buffer solution is not particularly limited as long as it exhibits buffering capacity, and at 25°C, a pH of 4 to 10 is preferred, and a pH of 5 to 9 is more preferred. In addition, in the immunoassay kit, the concentration of the buffer solution in the solution containing anti-C4BP antibody is not particularly limited as long as it exhibits buffering capacity, and a concentration of 1 to 1000 mM is preferred, and a concentration of 1 to 600 mM is more preferred. If the sample diluent and sample pretreatment solution contain buffer, the buffer concentration is preferably 50 to 600 mM, and if the detection reagent containing the detection substance contains buffer, the buffer concentration is preferably 1 to 100 mM.

[0063] [First Embodiment] The first embodiment of this model is an immunological assay kit that includes an anti-C4BP antibody and a detection substance in a single reagent.

[0064] In this embodiment, the concentration of the anti-C4BP antibody in the reagent containing the anti-C4BP antibody and the detection substance is not particularly limited as long as it is an amount that effectively suppresses nonspecific reactions. The concentration of the anti-C4BP antibody in the reagent is preferably 1 to 200 μg / mL, and more preferably 10 to 100 μg / mL.

[0065] In this embodiment, the type and amount of detection substances included in the immunological measurement kit can be appropriately selected depending on the immunological measurement method used.

[0066] [Second Embodiment] An immunological measurement kit according to a second embodiment of this model comprises a first reagent (R1) containing the anti-C4BP antibody and a second reagent (R2) containing the detection substance.

[0067] In this embodiment, the amount of anti-C4BP antibody contained in the first reagent is not particularly limited as long as it is sufficient to suppress non-specific reactions. The concentration of anti-C4BP antibody in the first reagent is preferably 1 to 200 μg / mL, and more preferably 1 to 50 μg / mL.

[0068] In this embodiment, the type and amount of detection substance contained in the second reagent can be appropriately selected depending on the immunological measurement method used. For example, if the immunological measurement method is the LTIA method, the second reagent preferably contains latex particles having an absorbance of 1 to 10 OD at 600 nm.

[0069] <Non-specific reaction inhibitor for immunoassays> A third aspect of the present invention is an immunological nonspecific reaction inhibitor comprising an anti-C4BP antibody. Because this immunological nonspecific reaction inhibitor comprises an anti-C4BP antibody, it can suppress nonspecific reactions in immunological measurements. In particular, it can effectively suppress nonspecific reactions when the sample contains C4BP.

[0070] Examples of immunological measurements in this embodiment include those described above.

[0071] The nonspecific reaction inhibitor for immunological measurement according to this embodiment is added to the sample in an amount that exerts the effect of suppressing nonspecific reactions. For example, the concentration of anti-C4BP antibody in the nonspecific reaction inhibitor for immunological measurement is preferably 10 to 5000 μg / mL, and more preferably 100 to 1000 μg / mL. Furthermore, in immunological measurement, it is preferable to mix the nonspecific reaction inhibitor for immunological measurement according to this embodiment with the sample so that the free C4BP content in the sample is 20 μg / mL or less. Here, "free C4BP" refers to C4BP that is not bound to anti-C4BP antibody. [Examples]

[0072] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0073] <Suppression of non-specific reaction in the LTIA method: Measurement of sIL-2R and C4BP> The non-specific reaction suppression effect was confirmed by the immunological measurement method of one aspect of the present invention. The confirmation of the non-specific reaction suppression effect was performed by comparing the measured value of interleukin 2 receptor (sIL-2R) in the specimen when no anti-C4BP antibody was used with the measured value of sIL-2R in the specimen when an anti-C4BP antibody was used. Human serum specimens 1 to 2 were used as specimens.

[0074] [Reference Example 1] Measurement by ELISA method 1. Measurement method 1-1. Measurement reagent An anti-sIL-2R monoclonal antibody was used as the measurement reagent. The anti-sIL-2R monoclonal antibody was obtained by a method well known to those skilled in the art using sIL-2R antigen as an immunogen. 1-2. Specimen Specimen 1 was used as the specimen.

[0075] 1-3. sIL-2R measurement procedure The method for measuring the content of sIL-2R contained in Specimen 1 by ELISA is described below. The anti-sIL-2R monoclonal antibody was dissolved in PBS so that the content of the anti-sIL-2R monoclonal antibody per 1 mL of PBS was 10 μg / mL. 100 μL of the obtained solution was dispensed into each well of a 96-well microplate and allowed to stand at room temperature overnight.

[0076] Each well was washed three times with 400 μL of PBST, and then 200 μL of BSA-PBST was added to each well for blocking at room temperature for 1 hour. This was used as an ELISA plate.

[0077] After washing each well of the ELISA plate three times with 400 μL of PBST, 100 μL of sample 1 or sample 2, diluted 40-fold with BSA-PBST, was added to each well and allowed to stand at room temperature for 1 hour.

[0078] After washing each well three times with 400 μL of PBST, 100 μL of biotin-labeled anti-sIL-2R monoclonal antibody diluted to 0.50 μg / mL with BSA-PBST was dispensed into each well and allowed to stand at room temperature for 1 hour.

[0079] After washing each well three times with 400 μL of PBST, 100 μL of HRP-labeled streptavidin (ThermoFishier 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.

[0080] After washing each well three times with 400 μL of PBST, 100 μL of citrate buffer (pH 5.0) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added, and the mixture was left in the dark at room temperature for 10 minutes. Then, 100 μL of 1.5N sulfuric acid was added to stop the enzymatic reaction, and the absorbance at a wavelength of 492 nm was measured. A standard substance with a known concentration was used as a calibrator, and the amount of sIL-2R contained in sample 1 was calculated.

[0081] 1-4. C4BP Measurement Procedure The C4BP content in sample 1 was measured using the ELISA Kit for C4 Binding Protein Alpha (Cloud-Clone Corp.) according to the manufacturer's instructions. Sample 1 was used after being diluted 10,000 times with PBS.

[0082] 2.Measurement results The measurement results for Reference Example 1 are shown in Table 1.

[0083] [Comparative Example 1] Measurement by LTIA method 1.Measurement method 1-1. Measurement Reagents The following reagents, Reagent 1 and Reagent 2, were used as the measurement reagents.

[0084] Reagent 1 It contains the following components (concentrations indicate concentrations in the reagent; the same applies hereinafter). • 100 mM Imidazole Buffer 500mM NaCl 1.0% BSA • 0.05% Proclin300

[0085] Reagent 2 5 mM Tris-HCl (pH 7.0) • Anti-sIL-2R monoclonal antibody-sensitized latex (2 types) Anti-sIL-2R monoclonal antibody-sensitized latex was prepared based on the method described in Japanese Patent Publication No. 2017-181377. Specifically, a 1.0% latex solution with an average particle size of 0.3 μm (5 mM Tris buffer (hereinafter referred to as Tris-HCl or simply Tris) (pH 8.5)) was mixed with an equivalent volume of 5 mM Tris-HCl (pH 8.5) to which an anti-sIL-2R monoclonal antibody solution diluted to 0.36 mg / mL was added and the mixture was stirred. Then, an equivalent volume of 0.5% BSA-containing 5 mM Tris-HCl (pH 8.5) was added and the mixture was stirred to prepare an anti-sIL-2R monoclonal antibody-sensitized latex particle solution. This latex particle solution was diluted with 5 mM Tris-HCl (pH 7.0) so that the absorbance at 600 nm was approximately 5.0 OD to prepare the second reagent.

[0086] 1-2. Specimen Sample 1 was used as the sample.

[0087] 1-3. sIL-2R Measurement Procedure The method for measuring the sIL-2R content in sample 1 using the LTIA method is described below. 120 μL of the first reagent was added to 5.6 μL of sample 1. The resulting solution was incubated at 37°C for 5 minutes, and then 40 μL of the second reagent was added and stirred to obtain the sample solution. The absorbance change due to the aggregation of latex particles caused by the binding of sIL-2R, the substance to be measured, and the anti-sIL-2R antibody, the detection substance, in the sample solution was measured over 5 minutes at a primary wavelength of 570 nm and a secondary wavelength of 800 nm. The obtained absorbance change was then applied to a calibration curve obtained by measuring standard substances of known concentration, and the sIL-2R content was calculated. A Hitachi 7180 automatic analyzer was used to measure absorbance. 2.Measurement results The measurement results for Comparative Example 1 are shown in Table 1.

[0088] [Comparative Example 2] Measurement by LTIA method 1.Measurement method The measurement was performed in the same manner as in Comparative Example 1, except that the sample processing described below was performed before measurement.

[0089] 1-1. Sample Processing Sample 1 and diluent physiological saline (product name: Otsuka Saline Injection, manufactured by Otsuka Pharmaceutical Co., Ltd.) were mixed in a 1:1 ratio. The mixed solution was left to stand at 4°C for 18 hours, and then centrifuged at 2130G for 10 minutes.

[0090] 2.Measurement results The measurement results for Comparative Example 2 are shown in Table 1.

[0091] [Example 1] 1.Measurement method The measurement was performed in the same manner as in Comparative Example 1, except that the sample processing described below was performed before measurement.

[0092] 1-1. Sample Processing Sample 1 and an anti-C4BP antibody solution (Proteintech, catalog number: 11819-1-AP, content 500 μg / mL) were mixed in a 1:1 ratio. The mixed solution was allowed to stand at 4°C for 18 hours, and then centrifuged at 2130 G for 10 minutes. The supernatant after centrifugation was used for measurement. The anti-C4BP antibody concentration in the sample solution at the stage of measuring the absorbance change due to the aggregation of latex particles was 8.5 μg / mL.

[0093] 2.Measurement results The measurement results for Example 1 are shown in Table 1.

[0094] [Reference Example 2] Measurement by ELISA method 1.Measurement method The measurement was performed in the same manner as in Reference Example 1, except that Sample 2 was used as the sample. 2.Measurement results The measurement results for Reference Example 2 are shown in Table 2.

[0095] [Comparative Example 3] Measurement by LTIA method 1.Measurement method The measurement was performed in the same manner as in Comparative Example 1, except that Sample 2 was used as the sample. 2.Measurement results The measurement results for Comparative Example 3 are shown in Table 2.

[0096] [Comparative Example 4] Measurement by LTIA method 1.Measurement method The measurement was performed in the same manner as in Comparative Example 2, except that Sample 2 was used as the sample. 2.Measurement results The measurement results for Comparative Example 4 are shown in Table 2.

[0097] [Example 2] Measurement by LTIA method 1.Measurement method The measurement was performed in the same manner as in Example 1, except that Sample 2 was used as the sample. 2.Measurement results The measurement results for Example 2 are shown in Table 2.

[0098] In Tables 1 and 2 below, "Sample Processing" indicates that the sample was processed using the method described above.

[0099] In Tables 1 and 2 below, the values ​​shown as converted values ​​(U / mL) are calculated by multiplying the measured values ​​(U / mL) for Examples 1-2, Comparative Example 2, and Comparative Example 4 by 2, which is the dilution factor of the sample at the time of sample processing. For Examples 1-2, Comparative Example 2, and Comparative Example 4, since Sample 1 or Sample 2 was measured after being diluted twice by the sample processing described above, the converted value (U / mL) was calculated by multiplying the diluted measurement value by the dilution factor to convert it back to the original solution concentration.

[0100] In Tables 1 and 2 below, "vs. ELISA (%)" indicates the ratio of the measured value (U / mL) or converted value (U / mL) of each experimental example to the measured value (U / mL) of Reference Example 1 or Reference Example 2, which is set to 100%. Here, measured value (U / mL) is used for experimental examples where sample processing was not performed, and converted value (U / mL) is used for experimental examples where sample processing was performed. For example, in Table 1 below, the percentage of the measured value (U / mL) in Comparative Example 1 and the percentage of the converted values ​​(U / mL) in Comparative Example 2 and Example 1 are shown as ELISA (%) relative to the measured value (U / mL) in Reference Example 1, which is set to 100%. For example, in Table 2 below, the percentage of the measured value (U / mL) in Comparative Example 3 and the percentage of the converted values ​​(U / mL) in Comparative Example 4 and Example 2 are shown as ELISA (%) relative to the measured value (U / mL) in Reference Example 2, which is set to 100%.

[0101] [Table 1]

[0102] [Table 2]

[0103] In Table 1, the ELISA ratio (%) for Comparative Example 1 was 134.0%. The ELISA method used in Reference Example 1 is a heterogeneous method that performs (B) / (F) separation and is less susceptible to nonspecific reactions. In contrast, the LTIA method used in Comparative Example 1 is a homogeneous method that does not perform (B) / (F) separation, and it is thought that the nonspecific binding of C4BP to sIL-2R or anti-sIL-2R antibody resulted in a higher measured value (U / mL) compared to Reference Example 1.

[0104] In Table 1, the ELISA ratio (%) for Comparative Example 2 was 118.6%, and similar to Comparative Example 1, the measured value (U / mL) was higher than that of Reference Example 1. This indicates that non-specific reactions caused by C4BP are not suppressed when sample processing is performed using a liquid that does not contain anti-C4BP antibodies.

[0105] In Table 1, the ELISA ratio (%) for Example 1 was 95.2%, which was lower than the measured values ​​(U / mL) for Comparative Examples 1 and 2. This indicates that the addition of anti-C4BP antibody to the sample suppressed the non-specific reaction caused by C4BP.

[0106] Table 1 shows that the C4BP content of Example 1 was 16.4 μg / mL, which was lower than the C4BP content of Reference Example 1 (43.5 μg / mL). This indicates that adding anti-C4BP antibody to the sample causes the anti-C4BP antibody to bind to C4BP, reducing the amount of free C4BP detectable by ELISA for C4BP measurement.

[0107] In Example 2, a different sample was used than in Example 1. Specifically, in Sample 1 used in Example 1, the LTIA measurement value was higher than the ELISA measurement value, whereas in Sample 2 used in Example 2, the ELISA measurement value and the LTIA measurement value were similar. Furthermore, the C4BP content in Sample 1 was high at 43.5 μg / mL, while the C4BP content in Sample 2 was 9.7 μg / mL.

[0108] In Table 2, there was no significant difference between the measured values of Reference Example 2 and the measured values or converted values of Comparative Examples 3-4 and Example 2. This indicated that the influence of the anti-C4BP antibody on the measured values derived from the measurement target substance was minor. The C4BP content contained in Specimen 2 used in Table 2 was lower than the C4BP content contained in Specimen 1 used in Table 1. Therefore, when the C4BP content contained in the specimen was low, it was considered that the possibility of non-specific reaction caused by C4BP was low.

[0109] <Suppression of Non-specific Reaction in the LTIA Method: Measurement of PCT> The non-specific reaction suppression effect was confirmed by the immunological measurement method of one aspect of the present invention. The confirmation of the non-specific reaction suppression effect was performed by comparing the measured value of procalcitonin (PCT) in the specimen when the anti-C4BP antibody was not used with the measured value of PCT in the specimen when the anti-C4BP antibody was used. As specimens, human serum specimens 3-7 of multiple persons (5 persons) were used. Specimens 3 and 4 (deviating specimens) exhibited non-specific reactions, and the measured values by the LTIA method were significantly different from the measured values by the LBA (Liquid-phase Binding Assay) method (Reference Example 3 described later). Specimens 5-7 (control specimens) were specimens in which the measured values by the LTIA method showed values close to the measured values by the LBA method (Reference Example 3 described later).

[0110] [Reference Example 3] Measurement by the LBA Method 1. Measurement Method 1-1. Measurement Reagent As the measurement reagent, Mutaswako (registered trademark) PCT (FUJIFILM Wako Pure Chemical Corporation) was used. 1-2. Specimen As specimens, specimens 3-7 were used.

[0111] 1-3. Measurement Procedure Measurement was performed using Mutaswako (registered trademark) i30 (FUJIFILM Wako Pure Chemical Corporation) according to the attached document of the measurement reagent.

[0112] 2.Measurement results The measurement results for Reference Example 3 are shown in Table 3.

[0113] [Comparative Example 5] Measurement by LTIA method 1.Measurement method 1-1. Measurement Reagents The following reagents, Reagent 1 and Reagent 2, were used as the measurement reagents.

[0114] Reagent 1 It contains the following components (concentrations indicate concentrations in the reagent; the same applies hereinafter). • 100 mM Bis-Tris-HCl (pH 6.5) 600mM NaCl 0.2% BSA

[0115] Reagent 2 5 mM MOPS-NaOH (pH 7.0) • Anti-human PCT monoclonal antibody-sensitized latex (2 types) The anti-human PCT monoclonal antibody was obtained using a commercially available PCT antigen by a method well known to those skilled in the art. A 1.0% latex solution with an average particle size of 0.3 μm (5 mM Tris buffer (hereinafter referred to as Tris-HCl or simply Tris) (pH 8.5)) was mixed with an equivalent volume of 5 mM Tris-HCl (pH 8.5) to prepare an anti-human PCT monoclonal antibody solution diluted to 0.36 mg / mL. Then, an equivalent volume of 0.5% BSA-containing 5 mM Tris-HCl (pH 8.5) was added and mixed to prepare an anti-human PCT monoclonal antibody-sensitized latex particle solution. This latex particle solution was diluted with 5 mM MOPS-NaOH (pH 7.0) to an absorbance of approximately 5.5 OD at 600 nm to prepare the second reagent.

[0116] 1-2. Sample Processing Samples 3-7 were mixed with PBS buffer (pH 7.4) in a 1:1 ratio and then subjected to measurement.

[0117] 1-3. Measurement Procedure Each sample was mixed with the first and second reagents, and the PCT concentration in the sample was measured using a Hitachi 3500 automated analyzer. Specifically, 120 μL of the first reagent was added to 12 μL of the sample and incubated at 37°C for 5 minutes. Then, 40 μL of the second reagent was added and stirred to obtain the sample solution. The absorbance change due to the binding and aggregation of PCT, the substance to be measured, and anti-human PCT monoclonal antibody, the detection substance, in the sample solution was measured over a 5-minute period at a primary wavelength of 570 nm and a secondary wavelength of 800 nm. The amount of absorbance change was applied to a calibration curve obtained by measuring standard substances of known concentration, and the measured value was calculated.

[0118] 2.Measurement results The measurement results for Comparative Example 5 are shown in Table 3.

[0119] [Comparative Example 6] Measurement by LTIA method: HBR added to the sample 1.Measurement method The measurement was performed in the same manner as in Comparative Example 5, except that the sample processing described below was performed before measurement instead of the sample processing in Comparative Example 5.

[0120] 1-1. Sample Processing PBS buffer (pH 7.4) was mixed with HBR (SCANTIBODIES Laboratory), a commercially available non-specific reaction inhibitor, to a concentration of 0.5 mg / mL. Samples 3-7 were mixed with the PBS buffer containing HBR in a 1:1 ratio for measurement. The anti-C4BP antibody concentration in the sample solution at the stage of measuring the absorbance change due to the aggregation of latex particles was 17.4 μg / mL.

[0121] 2.Measurement results The measurement results for Comparative Example 6 are shown in Table 3.

[0122] [Example 3] Measurement by LTIA method: Anti-C4BP antibody added to the sample. 1.Measurement method The measurement was performed in the same manner as in Comparative Example 6, except that an anti-C4BPA polyclonal antibody solution (0.5 mg / mL, Invitrogen) was mixed with the sample instead of a 0.5 mg / mL HBR solution.

[0123] 2.Measurement results The measurement results for Example 3 are shown in Table 3.

[0124] In Table 3 below, entries marked "Sample Processing" indicate that the sample was processed using the method described above.

[0125] In Table 3 below, the values ​​shown as measured values ​​(ng / mL) are the actual measured values ​​in Reference Example 3, and the values ​​calculated by multiplying the actual measured values ​​by 2, which is the sample dilution ratio at the time of sample processing, in Examples 3 and Comparative Examples 5-6. For Example 3 and Comparative Examples 5-6, since Sample 1 or Sample 2 was measured after being diluted twice by the sample processing described above, the converted value (ng / mL) was calculated by multiplying the diluted measurement value by the dilution factor to convert it back to the original concentration.

[0126] [Table 3]

[0127] Table 3 shows that the measured values ​​for samples 5-7 (control samples) were generally equivalent to those for Reference Example 3, Comparative Examples 5-6, and Example 3. This indicates that the addition of anti-C4BP polyclonal antibody does not affect the measured values ​​of samples that do not exhibit a nonspecific reaction.

[0128] Table 3 shows that the measured value of sample 3 (deviant sample) was less than 0.02 ng / mL in Reference Example 3. In Comparative Example 5, it was 0.80 ng / mL, which deviated from the measured value of Reference Example 3. On the other hand, the measured value of Example 3 was 0.68 ng / mL, showing a tendency to approach the measured value of Reference Example 1. Table 3 shows that the measured value of sample 4 (deviant sample) was 0.05 ng / mL in Reference Example 1. In Comparative Example 5, it was 1.22 ng / mL, which deviated from the measured value in Reference Example 3. In Comparative Example 6, it was 2.12 ng / mL, which deviated even more significantly than the measured value in Comparative Example 3. On the other hand, the measured value of Example 3 was 0.98 ng / mL, showing a tendency to approach the measured value in Reference Example 1. This demonstrated that using anti-C4BP polyclonal antibodies in immunoassay methods suppresses the influence of non-specific reactions originating from the sample.

Claims

1. An immunoassay method for measuring target substances contained in a sample, A step of bringing the sample into contact with the anti-C4BP antibody. A step of bringing a detection substance that specifically binds to the substance to be measured into contact with the sample, and The process includes measuring a signal corresponding to the content or titer of the substance to be measured, The immunological measurement method described above.

2. The aforementioned sample contains C4BP at a concentration of 20 μg / mL or higher. The immunological measurement method according to claim 1.

3. The anti-C4BP antibody is a polyclonal antibody. The immunological measurement method according to claim 1.

4. The anti-C4BP antibody is an anti-C4BPα antibody. The immunological measurement method according to claim 1.

5. The step of bringing the sample and the anti-C4BP antibody into contact is This includes adjusting the concentration of free C4BP, which is not bound to the anti-C4BP antibody, in the sample to 20 μg / mL or less. The immunological measurement method according to claim 1.

6. The step of bringing the sample and the anti-C4BP antibody into contact, The step of bringing the detection substance, which specifically binds to the substance to be measured, into contact with the sample, Perform these actions simultaneously or in this order. The immunological measurement method according to claim 1.

7. The immunoassay method is a homogeneous assay. The immunological measurement method according to claim 1.

8. Anti-C4BP antibody, A detection substance that specifically binds to the substance to be measured, Immunological measurement kit.

9. The first reagent containing the anti-C4BP antibody, A second reagent containing the aforementioned detection substance, The immunoassay kit according to claim 8.

10. The first reagent contains the anti-C4BP antibody, It is contained at a concentration of 1 to 200 μg / mL. The immunoassay kit according to claim 9.

11. A non-specific reaction inhibitor for immunoassays, containing an anti-C4BP antibody.

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