Method for measuring fragments containing the 7S domain of human type IV collagen and kit for use therein

Abstract

A method for measuring a fragment including 7S-domain of human type-IV collagen in a sample through sandwich immunoassay, by using: a capture antibody obtained by immobilizing, on a carrier, a first monoclonal antibody capable of specifically binding to 7S-domain of human type-IV collagen; and a labelled antibody obtained by binding, to a labeling substance, a second monoclonal antibody capable of specifically binding to 7S-domain of human type-IV collagen.

Description

[Technical field]

[0001] The present invention relates to a method for measuring a fragment containing the 7S domain of human type IV collagen in a sample and a kit for use in said method. [Background technology]

[0002] Type IV collagen is a major protein that constitutes the basement membrane together with laminin, and its structural unit is a trimer having a helical structure consisting of three α chains. Although the subunits that constitute the trimer differ depending on the tissue, in many tissues including the liver, the trimer is composed of two α1 chains and one α2 chain. Type IV collagen molecule has a structure specific to type IV collagen molecule at both ends of the TH domain (Triple Helical collagenous domain) that forms the helical structure, and the structure of the N-terminus is called the 7S domain, and the structure of the C-terminus is called the NC1 domain (Noncollagenous domain). The NC1 domain at the C-terminus forms a dimer with the NC1 domain of another type IV collagen molecule, and the 7S domain at the N-terminus forms a tetramer with the 7S domain of another type IV collagen molecule. All of these multimers are firmly bound to each other by covalent bonds, and the type IV collagen molecule forms a mesh structure (Chicken-wire network) by polymerization of these.

[0003] In human liver, hepatic fibrosis gradually progresses to liver cirrhosis or liver cancer due to chronic hepatitis B or C, chronic alcoholic hepatitis, or progressive non-alcoholic steatohepatitis (NASH), and it is known that type IV collagen increases at this time. As liver fibrosis progresses, the amount of type IV collagen leaking into the blood from the basement membrane increases, so the blood concentration of components derived from type IV collagen molecules is an indicator of the progression of liver fibrosis. In particular, the 7S domain, which is tightly bound by a covalent bond, is thought to be resistant to the effects of proteolytic enzymes and stable in the blood, and is used as a biomarker for the progression (severity) of liver fibrosis and the diagnosis of liver cirrhosis.

[0004] However, the only measurement reagent for the 7S domain approved as a diagnostic agent is the "Type IV Collagen 7S Kit (manufactured by DENIS Pharma Co., Ltd.)," which uses radioimmunoassay (RIA). The Type IV Collagen 7S Kit is a reagent that uses anti-human type IV collagen rabbit polyclonal antibody.

[0005] Furthermore, Japanese Patent Application Laid-Open No. 2-1553 (Patent Document 1) describes a method for quantifying human type IV collagen 7S domain by a one-step sandwich immunoassay, in which a monoclonal antibody that cross-reacts with pepsin-solubilized human type IV collagen 7S domain is used as an enzyme-labeled antibody, and a monoclonal antibody that reacts only with human type IV collagen 7S domain is bound to a solid support to form an antibody-bound solid support (capture antibody). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 2-1553 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the above-mentioned type IV collagen 7S kit uses a polyclonal antibody against human type IV collagen and is based on the principle of competitive binding inhibition, so the measurement accuracy is still insufficient. In particular, the inventors have found that there is a problem that the measurement value is high in samples from healthy subjects, resulting in a high false positive rate, making it unsuitable for screening and differentiation of patients in the early stages of liver fibrosis.

[0008] Furthermore, the present inventors have found that, while various types of collagen fragments, ranging from small to large molecules, are present in the blood from patients with liver fibrosis (patients with cirrhosis, NASH, etc.) as type IV collagen fragments containing the 7S domain, it is difficult to comprehensively measure all of these collagen fragments using the type IV collagen 7S kit or the method described in Patent Document 1, which uses a monoclonal antibody that cross-reacts with the pepsin-solubilized human type IV collagen 7S domain as an enzyme-labeled antibody, because small molecular collagen fragments cannot be detected sufficiently, and as a result, there is a problem that the measurement accuracy may decrease depending on the sample.

[0009] Furthermore, the inventors have found that these conventional methods also have the problem that the measurement accuracy is easily reduced due to the influence of sites other than the 7S domain of type IV collagen and other components contained in the sample.

[0010] The present invention has been made in consideration of the problems newly discovered by the inventors described above, and aims to provide a measurement method that can reduce non-specificity for samples derived from healthy individuals (negative samples) without reducing specificity for samples derived from patients (positive samples), and can measure fragments containing the human type IV collagen 7S domain in a sample with high accuracy, and a kit for use therein. [Means for solving the problem]

[0011] As a result of intensive research to solve the above problems, the present inventors have found that a sandwich immunoassay combining a monoclonal antibody capable of site-specific binding to the 7S domain of human type IV collagen can sufficiently reduce the measurement value of a sample derived from a healthy person (negative sample) without reducing the measurement value of a sample derived from a patient (positive sample), and can achieve a higher measurement accuracy than before, particularly in the low concentration range of a fragment containing the 7S domain of human type IV collagen. In addition, this method can comprehensively measure any collagen fragment as a type IV collagen fragment containing the 7S domain, and further, it has been found that a higher measurement accuracy than before can be achieved by being less susceptible to the influence of substances present in the sample and sites other than the 7S domain of type IV collagen, thereby completing the present invention.

[0012] The present invention, based on these findings, has the following features. [1] A method for measuring a fragment containing the human type IV collagen 7S domain in a sample by sandwich immunoassay using a capture antibody in which a first monoclonal antibody capable of specifically binding to the human type IV collagen 7S domain is immobilized on a carrier, and a labeled antibody in which a second monoclonal antibody capable of specifically binding to the human type IV collagen 7S domain is bound to a labeling substance. [2] a capture step of contacting the sample with the capture antibody and capturing the fragment containing the human type IV collagen 7S domain with the capture antibody; a labeling step of contacting the labeled antibody with the fragment containing the human IV collagen 7S domain captured by the capture antibody after the capture step, thereby labeling the fragment containing the human IV collagen 7S domain captured by the capture antibody; The measurement method according to [1], comprising: [3] The method according to [1] or [2], wherein the salt concentration of the reaction system between the fragment containing the human type IV collagen 7S domain and the first monoclonal antibody is 0.35 M or more. [4] A kit for use in the measurement method according to any one of claims 1 to 3, A first monoclonal antibody capable of specifically binding to the 7S domain of human type IV collagen, and a carrier capable of binding to the first monoclonal antibody; a second monoclonal antibody capable of specifically binding to the 7S domain of human type IV collagen; and a labeling substance capable of binding to the second monoclonal antibody; Including the kit. [5] A capture antibody in which a first monoclonal antibody is immobilized on the carrier; a labeled antibody in which a second monoclonal antibody is bound to the label; The kit according to [4], Effect of the Invention

[0013] According to the present invention, it is possible to provide a measurement method that can reduce non-specificity for samples derived from healthy individuals (negative samples) without reducing specificity for samples derived from patients (positive samples), and that can measure fragments containing the human type IV collagen 7S domain in a sample with high accuracy, and a kit for use therein. [Brief description of the drawings]

[0014] [Figure 1] 1 is an electrophoretic photograph showing the results of SDS-PAGE in the collagenase treatment of Example 1(1). [Diagram 2] 1 is a graph showing the relationship between the concentration of each monoclonal antibody and absorbance when type IV collagen 7S fragment is used as an antigen. [Diagram 3] 1 is a graph showing the relationship between the concentration of each monoclonal antibody and absorbance when a pepsin-solubilized type IV collagen fragment was used as an antigen. [Figure 4] 1 is a graph showing the relationship between the concentration of type IV collagen 7S fragment in a standard solution and the amount of luminescence. [Diagram 5] 1 is a graph showing the relationship between the actual concentration of type IV collagen 7S fragment in a standard solution and the coefficient of variation of the quantitative value. [Figure 6] FIG. 1 is a scatter diagram showing the correlation between the measurement results (CL 7S) of Example 2 and the measurement results (RIA 7S) of Comparative Example 1. [Figure 7] FIG. 13 is a scatter diagram showing the correlation between the measurement results of Example 2 (CL 7S) and the measurement results of Comparative Example 1 (RIA 7S) for the healthy subject group only. [Figure 8] 1 is a graph showing the distribution of measured values ​​for healthy subject group 1 in Example 3 ((b) CL 7S) and Comparative Example 2 ((a) RIA 7S). [Figure 9] 1 is a graph showing the distribution of measured values ​​for healthy subject group 2 in Example 3 ((b) CL 7S) and Comparative Example 2 ((a) RIA 7S). [Figure 10] FIG. 13 is a diagram showing ROC curves for Example 2 (CL 7S) and Comparative Example 1 (RIA 7S) for the cirrhosis group. [Figure 11] FIG. 13 shows ROC curves for Example 2 (CL 7S) and Comparative Example 1 (RIA 7S) for the NASH group. [Figure 12] 1 is a graph showing the amount of luminescence of each fraction of sample LC58 (a), the amount of luminescence of each fraction of sample LC51 (b), and the amount of luminescence of each fraction of healthy subject samples (c) when CIV09 antibody-immobilized magnetic particles are used. [Figure 13] 1 is a graph showing the luminescence intensity of each fraction of sample LC58 (a) and the luminescence intensity of each fraction of type IV collagen 7S fragment (b) when CIV09 antibody-immobilized magnetic particles or CIV03 antibody-immobilized magnetic particles are used. [Figure 14] 1 is a graph showing the amount of luminescence in each fraction of sample LC51 when CIV09 antibody-immobilized magnetic particles or CIV03 antibody-immobilized magnetic particles are used. [Figure 15] 15 is a graph showing the quantitative values ​​(pool recovery values) of each of pools 1 to 6 in FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described in detail below based on preferred embodiments thereof.

[0016] <Method for measuring fragments containing the 7S domain of human type IV collagen> The present invention provides a method for measuring fragments containing the human type IV collagen 7S domain (hereinafter sometimes simply referred to as the "measurement method of the present invention") in which fragments containing the human type IV collagen 7S domain in a sample are measured by sandwich immunoassay using a capture antibody in which a first monoclonal antibody capable of specifically binding to the human type IV collagen 7S domain is immobilized on a carrier, and a labeled antibody in which a second monoclonal antibody capable of specifically binding to the human type IV collagen 7S domain is bound to a labeling substance.

[0017] In the present invention, the "sample" is not particularly limited as long as it is a sample in which a fragment containing the human type IV collagen 7S domain may be present. In general, a blood sample collected from a subject (human) is used, and the blood sample may be serum, plasma, or whole blood, preferably serum or plasma, and more preferably serum. The sample may be appropriately diluted or suspended in the following diluent as necessary, or may be pretreated. Examples of the pretreatment include collagenase treatment.

[0018] (Fragment containing the 7S domain of human type IV collagen) In the sample (preferably a blood specimen), various fragments of human type IV collagen molecules, from small to large molecules, may be present as components derived from human type IV collagen molecules. Examples of such fragments include a fragment consisting of a 7S domain that does not contain a TH domain and an NC1 domain (hereinafter, sometimes referred to as a "type IV collagen 7S fragment"); a fragment consisting of a TH domain that does not contain an NC1 domain and a 7S domain; a fragment consisting of an NC1 domain that does not contain a TH domain and a 7S domain; and a fragment consisting of two or more of the whole or part of these domains; and a complex of these fragments with other basement membrane components, such as laminin. In the present invention, the "7S domain" includes both a 7S domain that does not form a multimer and a 7S domain that forms a dimer to tetramer, the "TH domain" includes both a TH domain that does not form a helical structure and a TH domain that forms the helical structure, and the "NC1 domain" includes both an NC1 domain that does not form a multimer and an NC1 domain that forms a dimer.

[0019] In the present invention, the term "type IV collagen 7S fragment" refers to a gel filtration fraction of a degradation product obtained by further treating a pepsin-solubilized type IV collagen fragment solubilized by pepsin digestion with collagenase, which is preferably composed of a tetramer of the 7S domain, and has a molecular weight equivalent to that of the tetramer of the 7S domain of human type IV collagen, more specifically, a fragment contained in a gel filtration fraction having a molecular weight of 200,000 (or a mass of 200 kDa) or more and around 400,000 (for example, 300 kDa to 440 kDa), or a fragment of human type IV collagen containing the 7S domain having a molecular weight equivalent thereto. The time of the collagenase treatment varies depending on the concentration of collagenase added, but is preferably, for example, 30 minutes to 14 hours, and the treatment temperature is preferably 30 to 37°C. In the present invention, the term "pepsin-solubilized type IV collagen fragment" refers to a human type IV collagen fragment solubilized by pepsin digestion, and refers to a mixture containing two or more of the following: a fragment consisting of the 7S domain (type IV collagen 7S fragment), a fragment consisting of the TH domain, a fragment consisting of the NC1 domain, and a fragment containing two or more of the whole or part of each of these domains. The pepsin-solubilized type IV collagen fragment can be obtained by a conventionally known method as appropriate. For example, it can be obtained by treating an extract containing human type IV collagen molecules from human placenta with pepsin, and may be a commercially available product.

[0020] In the present invention, the "fragment containing the 7S domain of human type IV collagen" to be measured may be any fragment of human type IV collagen containing the 7S domain, such as the 7S fragment of type IV collagen; a fragment consisting of the 7S domain and all or part of the TH domain; a fragment consisting of the 7S domain, all or part of the TH domain, and the NC1 domain, and one of these may be used alone or a mixture of two or more of them. According to the measurement method of the present invention, the fragment containing the 7S domain of human type IV collagen can be measured whether it contains the 7S fragment of type IV collagen with a small molecular weight or a fragment with a large molecular weight in which the 7S domain may not be fully exposed, and the fragment containing the 7S domain of human type IV collagen can be measured with higher accuracy than conventional methods.

[0021] (Monoclonal antibody) In the present invention, the "first monoclonal antibody capable of specifically binding to the 7S domain of human type IV collagen" and the "second monoclonal antibody capable of specifically binding to the 7S domain of human type IV collagen" (hereinafter, in some cases, these are collectively referred to as "anti-7S domain antibody") are monoclonal antibodies capable of specifically recognizing and binding only to the 7S domain of human type IV collagen. In other words, the anti-7S domain antibody does not bind to any site other than the 7S domain of human type IV collagen. The fact that an antibody is the anti-7S domain antibody can be confirmed, for example, by the ability to specifically recognize and bind to the 7S fragment of type IV collagen; or, by the ability to bind to a molecule contained in a fraction corresponding to the molecular weight of the 7S domain of human type IV collagen (preferably a tetramer) among gel filtration fractions of a sample, and by the ability to bind not only to type IV collagen fragments containing the TH domain or NC1 domain (for example, pepsin-solubilized type IV collagen fragments solubilized by pepsin treatment) but also to the 7S fragment of type IV collagen.

[0022] In addition, the "antibody" in the present invention includes not only a complete antibody but also an antibody fragment (e.g., Fab, Fab', F(ab') 2, Fv, single-chain antibodies, diabodies, etc.), and minibodies in which the variable regions of antibodies are combined.

[0023] The anti-7S domain antibody of the present invention can be produced by appropriately adopting and improving a conventionally known method, for example, a method for producing a monoclonal antibody using cells (hybridomas) obtained by fusing an antibody-producing cell with a myeloma cell (typically, the method by Kohler and Milstein (Kohler & Milstein, Nature, 256:495, 1975)).

[0024] Examples of the antibody-producing cells include spleen cells, lymph node cells, peripheral blood leukocytes, etc. of animals immunized with an immunogen (e.g., mice, rats, hamsters, rabbits, monkeys, goats, sheep, donkeys, camels, alpacas, chickens), and antibody-producing cells obtained by allowing an immunogen to act in a culture medium on the above-mentioned cells or lymphocytes previously isolated from an animal that has not been immunized can also be used.

[0025] In the present invention, the immunogen is not particularly limited as long as it is a fragment containing the human type IV collagen 7S domain, and examples thereof include the type IV collagen 7S fragment, the pepsin-solubilized type IV collagen fragment, and a fragment obtained by partially digesting the pepsin-solubilized type IV collagen with collagenase (corresponding to the "collagenase-partially digested type IV collagen fragment" in the Examples).

[0026] As the myeloma cells, various known cell lines can be used. The antibody-producing cells and the myeloma cells may be of different animal species origin, as long as they are fusible, but are preferably of the same animal species origin.

[0027] The hybridoma is obtained, for example, by cell fusion between spleen cells obtained from a mouse immunized with the immunogen and mouse myeloma cells. From the many hybridomas obtained, hybridomas producing monoclonal antibodies showing high reactivity to the IV collagen 7S fragment are screened, and if necessary, further epitope analysis of the monoclonal antibodies produced by the selected hybridomas is performed to identify clones producing monoclonal antibodies binding to the IV collagen 7S fragment, thereby obtaining hybridomas producing monoclonal antibodies (anti-7S domain antibodies) capable of specifically binding to the human IV collagen 7S domain. Such monoclonal antibodies can be obtained by culturing the hybridomas or from the ascites of a mammal to which the hybridomas have been administered.

[0028] Furthermore, the anti-7S domain antibody can also be produced by recombinant DNA techniques, for example, if DNA encoding the antibody can be obtained. Examples of the recombinant DNA techniques include cloning DNA encoding the antibody from hybridomas or the antibody-producing cells, incorporating it into an appropriate vector, and introducing it into host cells (e.g., mammalian cell lines, E. coli, yeast cells, insect cells, plant cells, etc.) to produce a recombinant antibody (e.g., PJ Delves, Antibody Production: Essential Techniques, 1997, WILEY; P. Shepherd and C. Dean Monoclonal Antibodies, 2000, OXFORD UNIVERSITY PRESS; Vandamme AM et al., Eur. J. Biochem. 192: 767-775, 1990). In expressing DNA encoding the above-mentioned antibody, DNA encoding the heavy chain or light chain may be separately incorporated into an expression vector and used to transform host cells, or DNA encoding the heavy chain and light chain may be incorporated into a single expression vector and used to transform host cells (for example, the method described in WO 94 / 11523).

[0029] The recombinant antibody can be obtained in a substantially pure and homogeneous form by culturing the host cells and isolating and purifying the antibody from the cultured host cells or from the culture medium. The recombinant antibody can be separated and purified by a method used for ordinary polypeptide purification. If a transgenic animal (e.g., cow, goat, sheep, pig, etc.) incorporating an antibody gene is produced using a transgenic animal production technique, it is also possible to obtain a large amount of monoclonal antibodies derived from the antibody gene from the milk of the transgenic animal.

[0030] In the present invention, the first monoclonal antibody and the second monoclonal antibody may be the same or different from each other. When the sample is a blood specimen, the human type IV collagen 7S domain usually forms a dimer to tetramer (preferably a tetramer) in the blood specimen, so that even if the first monoclonal antibody and the second monoclonal antibody are the same type of antibodies that recognize and bind to the same site, each of them can bind to any one of the human type IV collagen 7S domains in the multimer.

[0031] (capture antibody) In the measurement method of the present invention, a capture antibody in which a first monoclonal antibody is immobilized on a carrier is used as a capture antibody for capturing the fragment containing the human type IV collagen 7S domain.

[0032] In the present invention, the "carrier" is not particularly limited as long as it can immobilize and support the first monoclonal antibody. The material of such a carrier is not particularly limited as long as it is generally used in immunoassays, and examples thereof include polymers (e.g., polystyrene, (meth)acrylic acid esters, polymethyl methacrylate, polyimide, nylon, etc.), gelatin, cellulose, nitrocellulose, glass, latex, silica, metals (e.g., gold, platinum, iron, cobalt, nickel, etc.), metal compounds (e.g., iron oxide, cobalt oxide, nickel ferrite, etc.), and composites and alloys thereof. In addition, the carrier may be surface-modified with one or more active groups selected from the group consisting of a carboxy group, an epoxy group, a tosyl group, an amino group, a hydroxy group, an isocyanate group, an azide group, an aldehyde group, a carbonate group, an allyl group, an aminooxy group, a maleimide group, a thiol group, and an NHS ester (N-hydroxy ester) group.

[0033] In the present invention, the shape of the carrier is not particularly limited and may be, for example, any of a plate, a fiber, a membrane, a particle, etc., but particles are preferred from the viewpoint of the tendency to easily control and uniformize the density of the first monoclonal antibody to be immobilized. Furthermore, the particles are preferably magnetic particles from the viewpoint of automation and shortening the time.

[0034] When the carrier is a particle, its size is not particularly limited, but the particle diameter is preferably within the range of 0.01 to 100 μm, and more preferably within the range of 0.1 to 10 μm. If the particle diameter of the particle is less than the lower limit, the magnetic force and time required for magnetic collection tend to be longer in the case of magnetic particles, while if the particle diameter exceeds the upper limit, the specific surface area of ​​the particle tends to be smaller, and the amount of the first monoclonal antibody bound to the particle tends to be smaller.

[0035] As such particles, conventionally known particles can be appropriately used, for example, known particles such as magnetic particles (ferrite-coated particles, carboxylated ferrite particles) described in JP-A-3-115862, and commercially available particles such as Dynabeads (manufactured by Thermo Fisher Scientific), Magnosphere (manufactured by JSR Corporation), Magrapid (manufactured by Sanyo Chemical Industries, Ltd.) can be appropriately used. Moreover, the particles may be one of these alone or a combination of two or more of them.

[0036] In the method for producing a capture antibody according to the present invention, the method for immobilizing the first monoclonal antibody on the carrier can be appropriately selected from conventionally known methods or methods based thereon, and the first monoclonal antibody may be immobilized directly or indirectly on the surface of the carrier. Examples of the direct binding method include a method in which the active group is imparted to the carrier, or a carrier having these active groups is used, and the first monoclonal antibody is bound to the carrier by covalent bonding via the active group.

[0037] As a method for indirect immobilization, for example, a substance that binds to the first monoclonal antibody is immobilized on the surface of the carrier, and the first monoclonal antibody is bound to the substance, thereby indirectly immobilizing the first monoclonal antibody on the surface of the carrier. The substance that binds to the first monoclonal antibody is not particularly limited, and examples thereof include a secondary antibody that can bind to the first monoclonal antibody, protein G, protein A, and a linker such as a linker molecule having the active group. In addition, when the first monoclonal antibody is biotinylated or avidinated (or streptavidinylated), the carrier may be avidinated (or streptavidinylated) or biotinylated, and the antibody may be immobilized by the interaction between biotin and avidin (or streptavidin).

[0038] Furthermore, the capture antibody used in the measurement method of the present invention may be prepared by first immobilizing the first monoclonal antibody directly or indirectly to a carrier capable of binding to the first monoclonal antibody, and then subjecting the capture antibody to a sandwich immunoassay. Alternatively, the capture antibody may be prepared by separately providing the first monoclonal antibody and a carrier capable of binding to the first monoclonal antibody to a reaction system for a sandwich immunoassay, and immobilizing the first monoclonal antibody to the carrier in the same reaction system, or by forming a complex between the first monoclonal antibody and a fragment containing the 7S domain of human type IV collagen and then immobilizing the first monoclonal antibody to the carrier. In this case, the combination of "a first monoclonal antibody and a carrier capable of binding to the first monoclonal antibody" may be any combination of the above-mentioned first monoclonal antibody and the carrier. When these are separately provided to the reaction system, however, the combination of the first monoclonal antibody and a carrier having a substance that binds to the first monoclonal antibody immobilized thereon, as described in the method for indirectly immobilizing the first monoclonal antibody to the carrier, and the combination of a biotinylated or avidinated (or streptavidinylated) first monoclonal antibody and an avidinated (or streptavidinylated) or biotinylated carrier are preferred.

[0039] The capture antibody according to the present invention contains at least the first monoclonal antibody and the carrier, and may further contain the linker, blocking agent, and the like, as necessary.

[0040] (labeled antibody) In the measurement method of the present invention, a labeled antibody in which a second monoclonal antibody is bound to a labeling substance is used as the labeled antibody for labeling the fragment containing the human type IV collagen 7S domain.

[0041] In the present invention, the "labeling substance" is not particularly limited as long as it can be bound to the second monoclonal antibody and detected, and examples thereof include enzymes; luminescent substances such as acridinium derivatives; fluorescent substances such as europium; fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE); 125 Examples of the labeling substance according to the present invention include radioactive substances such as I; low molecular weight labeling substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); gold particles; avidin; biotin; latex; dinitrophenyl (DNP); and digoxigenin (DIG). Among these, the labeling substance according to the present invention is preferably an enzyme or a luminescent substance, more preferably an enzyme, from the viewpoint of tending to enable a simple and stable test and being able to perform a simple, rapid and highly sensitive measurement. Examples of the enzyme include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, and luciferase.

[0042] In the method for producing the labeled antibody according to the present invention, the method for binding the labeling substance to the second monoclonal antibody can be appropriately selected from conventionally known methods or methods based thereon, and the binding may be direct or indirect. Examples of such binding methods include the same methods as those mentioned as the binding method between the carrier and the first monoclonal antibody in the capture antibody according to the present invention.

[0043] Furthermore, the labeled antibody used in the measurement method of the present invention may be prepared by first producing a labeled antibody in which a labeled substance capable of binding to the second monoclonal antibody is directly or indirectly bound to the second monoclonal antibody from a second monoclonal antibody and a labeled substance capable of binding to the second monoclonal antibody, and then subjecting the labeled antibody to a sandwich immunoassay. Alternatively, the labeled antibody may be prepared by separately providing a second monoclonal antibody and a labeled substance capable of binding to the second monoclonal antibody to a reaction system for a sandwich immunoassay, and then binding the labeled substance to the second monoclonal antibody in the same reaction system, or by forming a complex between the second monoclonal antibody and a fragment containing the human type IV collagen 7S domain and then binding the labeled substance to the second monoclonal antibody. In this case, the combination of "the second monoclonal antibody and a labeling substance capable of binding to the second monoclonal antibody" may be any combination of the above-mentioned second monoclonal antibody and the labeling substance, but when these are separately provided to the reaction system, a combination of the second monoclonal antibody and a labeling substance bound to a substance that binds to the second monoclonal antibody, and a combination of a biotinylated or avidinated (or streptavidinylated) second monoclonal antibody and an avidinated (or streptavidinylated) or biotinylated labeling substance are preferred. Examples of substances that bind to the second monoclonal antibody include the same substances as those listed as substances that bind to the first monoclonal antibody.

[0044] The labeled antibody according to the present invention contains at least the second monoclonal antibody and the labeling substance, and may further contain the linker, blocking agent, and the like, as necessary.

[0045] (Sandwich immunoassay) The measurement method of the present invention measures a fragment containing the human type IV collagen 7S domain in a sample by sandwich immunoassay using the capture antibody and the labeled antibody.

[0046] In the present invention, "measurement" includes detection of the presence or absence and amount of a target substance in a sample as a signal, as well as quantification or semi-quantification of the amount of the target substance. In the present invention, the target substance is preferably measured by detecting a signal generated by a labeling substance and quantifying the signal as necessary. The "signal" includes color development (coloring), reflected light, luminescence, fluorescence, radiation from a radioisotope, etc., and includes signals that can be confirmed by the naked eye as well as signals that can be confirmed by a measurement method or device depending on the type of signal.

[0047] A "sandwich immunoassay" is a method in which a target substance (in this invention, a "fragment containing the 7S domain of human type IV collagen") is captured by a capture antibody (in this invention, a "capture antibody") fixed (immobilized) on a solid phase, and the target substance is recognized by a detection antibody (in this invention, a "labeled antibody") to which a labeling substance is bound, forming a capture antibody-target substance-labeled antibody complex, which is then washed as necessary and then detected according to the type of labeling substance.

[0048] In the measurement method of the present invention, "capturing a target substance (in the present invention, "a fragment containing the human type IV collagen 7S domain") with a capture antibody (in the present invention, "capture antibody") immobilized (immobilized) on a solid phase" includes forming a complex between a first antibody (in the present invention, "first monoclonal antibody") that is not immobilized on a solid phase (in the present invention, "carrier") and the target substance, or forming a complex between a first antibody that is not immobilized on a solid phase, the target substance, and the labeled antibody (or the second antibody described below), and then immobilizing the first antibody that has formed the complex on the solid phase and capturing the target substance (or the target substance and the labeled antibody) on the solid phase via the first antibody. The combination of the first antibody and the solid phase in this case, including its preferred embodiment, is the same as the combination listed in "a first monoclonal antibody and a carrier capable of binding to the first monoclonal antibody" for the capture antibody described above.

[0049] Furthermore, in the measurement method of the present invention, "allowing a detection antibody bound to a labeling substance (in the present invention, "labeled antibody") to recognize a target substance" includes forming a complex between a second antibody (in the present invention, "second monoclonal antibody") not bound to a labeling substance and the target substance, or forming a complex between a second antibody not bound to a labeling substance, the target substance, and the capture antibody (or the above-mentioned first antibody), and then binding the labeling substance to the second antibody that formed the complex, and allowing the labeling substance to recognize the target substance (or the target substance and the capture antibody) via the second antibody. The combination of the second antibody and the labeling substance in this case, including its preferred embodiment, is the same as the combination listed in "second monoclonal antibody and a labeling substance capable of binding to the second monoclonal antibody" for the above-mentioned labeled antibody.

[0050] Examples of such sandwich immunoassays include the sandwich CLIA method, which is an embodiment of the chemiluminescent immunoassay (CLIA method), the sandwich CLEIA method, which is an embodiment of the chemiluminescent enzyme immunoassay (CLEIA method), the IRMA method (immunoradiometric assay), which is an embodiment of the radioimmunoassay (RIA method), the sandwich ECLIA method, which is an embodiment of the electrochemical immunoassay (ECLIA method), and the sandwich FIA method, which is an embodiment of the fluorescent immunoassay (FIA method).

[0051] In the measurement method of the present invention, the salt concentration of the reaction system between the fragment containing the human type IV collagen 7S domain and the first monoclonal antibody (preferably the reaction system in which the fragment containing the human type IV collagen 7S domain is captured by the capture antibody) is preferably 0.35 M (M: mol / L, the same applies below) or more. The salt concentration is more preferably 0.35 to 1.3 M, more preferably 0.42 to 1.0 M, and even more preferably 0.43 to 0.99 M. In particular, in the measurement method of the present invention, by having the salt concentration within the above range, non-specific reactions in samples derived from healthy subjects can be further reduced and measurement accuracy can be further improved.

[0052] The present inventors have found that, in the measurement of a fragment containing the 7S domain of human type IV collagen, by increasing the salt concentration during binding of the fragment with the first monoclonal antibody (preferably during capture with the capture antibody) to a level higher than the salt concentration (e.g., about 0.15 M) in conventional sandwich immunoassays, it is possible to attenuate the binding to the 7S domain of human type IV collagen contained in a sample derived from a healthy subject, and to maintain or enhance the binding to the 7S domain contained in a sample derived from a patient with liver fibrosis (such as a patient with cirrhosis or NASH). One of the reasons for this is considered to be that the composition of the collagen fragments containing the 7S domain of human type IV collagen contained in a sample derived from a patient is different from that of the collagen fragments contained in a sample derived from a healthy subject, and that the measurement method of the present invention reduces non-specificity to the collagen fragments contained in a sample derived from a healthy subject at a high salt concentration. In addition, the sample contains a wide variety of collagen fragments with different degrees of exposure of the 7S domain, including those with large molecular weights, depending on the length of the TH domains contained and the size of the collagen mesh structure. Therefore, at low salt concentrations, a discrepancy is likely to occur between the measured value using an anti-7S domain antibody and the actual amount of fragments containing the human type IV collagen 7S domain in the sample. However, by increasing the salt concentration, the mask is removed, exposing the 7S domain, which is thought to be one of the reasons why the discrepancy was eliminated.

[0053] The reaction system between the fragment containing the human IV collagen 7S domain and the first monoclonal antibody (preferably the reaction system for capturing with the capture antibody) is not particularly limited as long as it contains the sample, the first monoclonal antibody (preferably the capture antibody), and the salt, but is preferably an aqueous solution, and may further contain physiological saline, purified water, buffers such as MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, and glycine buffer, stabilizing proteins such as BSA, and various surfactants, and may further contain the labeled antibody when the reverse sandwich method or one-step method is adopted. Note that when these other components contain salt, the salt concentration of the reaction system also includes the concentration of the salt.

[0054] In addition, when the sample contains a salt, the salt concentration of the reaction system includes the concentration of the salt. A blood sample (whole blood, serum, plasma) generally contains a total of 140 mM to 150 mM of salt. For example, when a blood sample (whole blood, serum, plasma, etc.) is used as the sample, as in the following example, when 30 μL of a serum sample is added as a sample to 50 μL of a particle (capture antibody) suspension containing 0.6 M to 1.5 M of salt, the salt concentration of the reaction system in which the fragment containing the human type IV collagen 7S domain is captured by the capture antibody is 0.43 M to 0.99 M.

[0055] The salt is not particularly limited, but is preferably at least one selected from the group consisting of alkali metal ions, inorganic salts of alkali metal ions, and organic salts of alkali metal ions. Examples of the inorganic salts of alkali metal ions include chlorides of alkali metals, tartrates of alkali metals, nitrates of alkali metals, and sulfates of alkali metals. The alkali metal ions are preferably sodium ions and potassium ions. In this case, the salt concentration refers to a concentration equivalent to the concentration of the alkali metal ions.

[0056] In such a sandwich immunoassay, a first monoclonal antibody, a carrier capable of binding to the first monoclonal antibody (e.g., the capture antibody), the sample, and a second monoclonal antibody and a labeling substance capable of binding to the second monoclonal antibody (e.g., the labeled antibody) are contacted to finally form the complex (capture antibody-human type IV collagen 7S domain-containing fragment (target substance)-labeled antibody). Such a contact method is not particularly limited, and a conventionally known method or a method similar thereto can be appropriately adopted, for example, a method of adding a first monoclonal antibody and the carrier (e.g., the capture antibody), and / or a second monoclonal antibody and the labeling substance (e.g., the labeled antibody) to the sample; or, for example, a method of adding the sample, etc. to the capture antibody liquid (e.g., a particle suspension when the carrier is a particle) and / or the labeled antibody liquid. In addition, the sample may be diluted or suspended in a diluent as appropriate.

[0057] When the first monoclonal antibody, the carrier capable of binding to the first monoclonal antibody (e.g., the capture antibody), the sample, and the second monoclonal antibody, and the labeling substance capable of binding to the second monoclonal antibody (e.g., the labeled antibody) are brought into contact with each other, it is preferable that the salt concentration when the first monoclonal antibody (preferably the capture antibody) is brought into contact with the sample satisfies the above-mentioned condition, but other conditions are not particularly limited. For example, in addition to the solvent of the capture antibody liquid (e.g., a particle suspension medium when the carrier is a particle), the solvent of the labeled antibody liquid, and the diluent, a reaction buffer may be further added as appropriate. The solvent of the capture antibody liquid, the solvent of the labeled antibody liquid, the diluent, and the reaction buffer are not particularly limited, and examples thereof include, independently, physiological saline, purified water, and buffer solutions (sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.), and each may be independently added with a stabilizing protein such as BSA. When these solutions contain salt, the salt concentration of the reaction system includes the concentration of the salt. When the sample contains salt, the salt concentration of the reaction system includes the concentration of the salt.

[0058] In forming the complex, the content of the first monoclonal antibody in the reaction system (when two or more types are combined, the total amount of the antibodies, the same applies below) is not particularly limited and is adjusted appropriately depending on the type, concentration, detection method, etc. of the sample, but from the viewpoint of efficient capture in a short time, it is preferably, for example, 0.000001 to 0.01 w / v%, and more preferably 0.00001 to 0.001 w / v%. In this specification, the term "w / v%" refers to weight / volume percent (g / 100 mL).

[0059] Furthermore, in forming the complex, the content of the second monoclonal antibody in the reaction system (when two or more types are combined, the total amount of the antibodies; the same applies below) is not particularly limited and is adjusted appropriately depending on the type, concentration, detection method, etc. of the sample, but from the viewpoint of efficient labeling in a short period of time, it is preferably, for example, 0.000001 to 0.01 w / v%, and more preferably 0.00001 to 0.001 w / v%.

[0060] Furthermore, the conditions for the contact are not particularly limited and can be adjusted independently as appropriate. For example, the contact can be performed at 4 to 45°C, preferably 20 to 37°C; at a pH of about 6 to 9, preferably 6.5 to 8; for about 30 seconds to 12 hours, preferably 1 minute to 1 hour, but is not limited to these conditions.

[0061] Examples of the sandwich immunoassay include a two-step method, such as a forward sandwich method (a method in which capture by a carrier (preferably, a reaction between a capture antibody and a fragment containing the human type IV collagen 7S domain in a sample) and recognition by a labeling substance (preferably, a reaction between the fragment containing the human type IV collagen 7S domain bound to the capture antibody and the labeled antibody) are performed sequentially), a reverse sandwich method (a method in which recognition is performed in advance by a labeling substance (preferably, a labeled antibody is reacted with a fragment containing the human type IV collagen 7S domain in a sample), and the resulting complex is captured by a carrier (preferably, reacted with the capture antibody)), and a one-step method (a method in which the reaction of the capture antibody, the fragment containing the human type IV collagen 7S domain in a sample, and the labeled antibody is performed simultaneously in one step), and any of these can be used.

[0062] Among these, the measurement method of the present invention is preferably a two-step method, and more preferably a forward sandwich method, from the viewpoint of measurement accuracy. Examples of the measurement method of the present invention that is such a forward sandwich method include: a capture step of contacting the sample with the capture antibody and capturing the fragment containing the human type IV collagen 7S domain with the capture antibody; a labeling step of contacting the labeled antibody with the fragment containing the human IV collagen 7S domain captured by the capture antibody after the capture step, thereby labeling the fragment containing the human IV collagen 7S domain captured by the capture antibody; Examples of measurement methods include:

[0063] [Capturing process] In the capture step, the capture antibody is brought into contact with the sample, and the fragment containing the human IV collagen 7S domain is captured by the capture antibody through the binding between the fragment containing the human IV collagen 7S domain and the first monoclonal antibody, that is, a first immune complex is formed between the first monoclonal antibody and the fragment containing the human IV collagen 7S domain. The method and conditions for contacting the capture antibody with the sample are not particularly limited, but preferably satisfy the salt concentration condition, and the method and conditions described above for contacting the capture antibody, the sample, and the labeled antibody can be applied.

[0064] [Cleaning process] The measurement method of the present invention preferably further includes a washing step in which, after the capture step and before the labeling step described below, the fragment containing the human IV collagen 7S domain captured by the capture antibody is separated from other impurities not bound to (captured by) the capture antibody, and the impurities are removed. The method for removing the impurities is not particularly limited, and a conventionally known method or a method similar thereto can be appropriately adopted, for example, a method in which the capture antibody is recovered by centrifugation or magnetic collection after the capture step and the liquid phase (supernatant) is removed. In addition, in the washing step, injection and removal of a washing solution may be repeated as necessary. Examples of the washing solution include known neutral (preferably pH 6 to 9) buffer solutions (sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.), and may also be those to which a stabilizing protein such as BSA or a surfactant has been added.

[0065] [Labeling process] In the labeling step, the labeled antibody is contacted with the fragment containing the human IV collagen 7S domain captured by the capture antibody to label the fragment containing the human IV collagen 7S domain captured by the capture antibody, i.e., a second immune complex is formed between the second monoclonal antibody and the fragment containing the human IV collagen 7S domain captured by the capture antibody. The method and conditions for contacting the labeled antibody with the fragment containing the human IV collagen 7S domain captured by the capture antibody are not particularly limited, and the methods and conditions described above for contacting the capture antibody, the sample, and the labeled antibody can be applied.

[0066] The above-mentioned capture step and labeling step form a complex (second immune complex) containing the capture antibody-fragment containing the human type IV collagen 7S domain-labeled antibody. After the labeling step, a washing step may be included as necessary to remove the labeled antibody and other substances that did not form the complex. Such a washing step is the same as the above-mentioned washing step.

[0067] In the measurement method of the present invention, the labeling substance of the complex (capture antibody-fragment containing human type IV collagen 7S domain-labeled antibody) is measured by a predetermined method according to the labeling substance. For example, when the labeling substance of the labeled antibody is an enzyme, a chromogenic substrate, luminescent substrate, chemiluminescent substrate, etc. corresponding to the enzyme is added, and a signal (color development, luminescence, etc.) generated by reacting the enzyme with the substrate is detected. This allows the presence or absence and amount of the fragment containing human type IV collagen 7S domain in the sample to be detected as a signal. In addition, the amount of the fragment containing human type IV collagen 7S domain in the sample is generally quantified by comparison with the measured value using a standard solution containing a fragment containing human type IV collagen 7S domain at each concentration. The fragment containing human type IV collagen 7S domain used in the standard solution is not particularly limited, and examples thereof include the type IV collagen 7S fragment and the collagenase partially digested type IV collagen fragment. In this case, for example, the amount of the fragment containing human type IV collagen 7S domain in the sample can be determined by examining where the measured value obtained in the sample is located on a standard curve created based on the measured value using the standard solution.

[0068] The measurement method of the present invention can be used for diagnosis (evaluation of morbidity and risk) and support of diseases related to abnormal blood concentration of type IV collagen. Here, examples of "diseases related to abnormal blood concentration of type IV collagen" include diseases in which liver fibrosis progresses, such as chronic viral hepatitis type B and C, alcoholic hepatitis, nonalcoholic steatohepatitis (NASH), liver cirrhosis, and liver cancer.

[0069] In addition, the measurement method of the present invention can be used for diagnosing or assisting in or differentiating the progression (severity) of liver fibrosis, and for screening patients who may have liver fibrosis. In particular, the measurement method of the present invention can reduce non-specificity to samples from healthy subjects (negative samples) without reducing specificity to samples from patients (positive samples), and can achieve high measurement accuracy in the low concentration range of a fragment containing the human type IV collagen 7S domain, making it useful as a measurement method for screening patients with early stages of liver fibrosis from healthy subjects and for differentiating them from healthy subjects.

[0070] <Kit for use in the method for measuring fragments containing the 7S domain of human type IV collagen> The present invention relates to a kit for use in the measurement method of the present invention, A first monoclonal antibody capable of specifically binding to the 7S domain of human type IV collagen, and a carrier capable of binding to the first monoclonal antibody; a second monoclonal antibody capable of specifically binding to the 7S domain of human type IV collagen; and a labeling substance capable of binding to the second monoclonal antibody; The present invention also provides a kit for a method for measuring a fragment containing the 7S domain of human type IV collagen (hereinafter, sometimes simply referred to as the "kit of the present invention"). A capture antibody in which a first monoclonal antibody is immobilized on the carrier; a labeled antibody in which a second monoclonal antibody is bound to the label; It is preferable that the embodiment includes the following.

[0071] The first monoclonal antibody, the carrier capable of binding to the first monoclonal antibody, the second monoclonal antibody, the labeling substance capable of binding to the second monoclonal antibody, the capture antibody, and the labeled antibody contained in the kit of the present invention are as described in the measurement method of the present invention, including preferred embodiments. Also, the first monoclonal antibody, the carrier capable of binding to the first monoclonal antibody, the second monoclonal antibody, the labeling substance capable of binding to the second monoclonal antibody, the capture antibody, and the labeled antibody contained in the kit of the present invention may each independently be in a solid (powder) form or in a liquid form dissolved in a buffer solution or the like.

[0072] The kit of the present invention may further include components to be included in a typical sandwich assay such as ELISA, CLEIA, ECLIA, CLIA, FIA, immunochromatography, etc. For example, the kit may further include at least one selected from the group consisting of the standard solution (each concentration), the control reagent, the diluent, the solvent for the capture antibody solution (particle suspension medium, etc.), the solvent for the labeled antibody solution, the reaction buffer, the washing solution, and the dilution cartridge. In addition, for example, when the labeled substance is an enzyme, the kit may further include a substrate or a reaction stop solution necessary for detecting and quantifying the labeled substance. Furthermore, the kit may further include a pretreatment solution for pretreatment of the sample, an instruction manual for the kit, etc., as necessary. EXAMPLES

[0073] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples. In each example and comparative example, "%" indicates weight / volume percentage (w / v%: g / 100 mL) unless otherwise specified.

[0074] <Sandwich immunoassay using anti-type IV collagen 7S domain monoclonal antibody> Example 1 (1) Collagenase treatment The type IV collagen 7S fragment was prepared based on the method of Risteli et al. (Eur J Biochem, 108, p. 239-250). That is, first, a pepsin-solubilized type IV collagen fragment (derived from human placenta, Sigma, C-7521) prepared by pepsin digestion was dissolved in 0.5 M acetic acid, 0.2 M NaCl, 2 mM CaCl 2 The mixture was dialyzed against 50 mM Tris (pH 7.2) containing EDTA. Next, collagenase (Collagenase "Amano", Wako, 607-19021) was added and incubated at 37°C for 1 hour to further digest the pepsin-solubilized type IV collagen fragments (collagenase treatment). The supernatant obtained by centrifuging the collagenase-treated solution was further separated by gel filtration. A Superdex 200pg 16 / 60 gel filtration column and 1 mM EDTA 3Na and PBS were used as gel filtration buffers. FIG. 1 shows the results of SDS-PAGE (2-15% PAG Non-Reduced) of MW Marker (No. 1, molecular weight / 1000), pepsin-solubilized type IV collagen fragment (No. 2), collagenase treatment solution (No. 3) and its precipitate (No. 4) and supernatant (No. 5), and each fraction (fraction (GF Fr.) 3-7; No. 6-10). Fractions with molecular weights of 250,000 (or masses of 250 kDa) or more and around 400,000 (300 kDa to 440 kDa), which correspond to the molecular weight of the tetramer of human type IV collagen 7S domain, were collected and used as the type IV collagen 7S fragment described below.

[0075] In the present embodiment and comparative examples, the "type IV collagen 7S fragment" is a human type IV collagen fragment consisting of the 7S domain, not including the TH domain, and was obtained by treating with collagenase and selecting a gel filtration fraction. In addition, a collagenase partially digested type IV collagen fragment was used as the immunogen described below. The "collagenase partially digested type IV collagen fragment" is a fragment prepared by digesting a pepsin-solubilized type IV collagen fragment (manufactured by SIGMA) with collagenase at a treatment temperature of 25°C for 1 hour, the main component of which is a collagen fragment having a molecular weight of 500k or more, and is a mixture mainly consisting of the type IV collagen 7S fragment and a human type IV collagen fragment consisting of a part of the 7S domain and a part of the TH domain. The above-mentioned "pepsin-solubilized type IV collagen fragment" is a fragment prepared by solubilizing with pepsin digestion, and is a mixture of human type IV collagen fragment consisting of the 7S domain, the TH domain, or the NC1 domain, and human type IV collagen fragments containing two or more of the whole or part of each of these.

[0076] (2) Preparation of monoclonal antibodies A monoclonal antibody was produced using the "collagenase partially digested type IV collagen fragment" prepared in (1) above as an immunogen. Specifically, BALB / c mice were intraperitoneally immunized twice with 10 μg (1 mg / mL) of collagenase partially digested type IV collagen fragment mixed with an equal amount of complete Freund's adjuvant at an interval of two weeks. Furthermore, the mice were intraperitoneally immunized twice with incomplete Freund's adjuvant at an interval of two weeks. Furthermore, 10 μg of collagenase partially digested type IV collagen fragment dissolved in PBS was administered into the tail vein as the final immunization. Three days after the final immunization, the spleen was removed from the mouse, disaggregated into individual cells, and washed three times with RPMI-1640 medium. Logarithmic growth phase mouse myeloma cell line Sp2 / OAg14 was washed three times with RPMI-1640 medium, mixed with the spleen cells, and cell fusion was performed. The fused cells obtained were centrifuged (200×g, 5 min) to remove PEG, then suspended in RPMI-1640 medium containing 10% fetal bovine serum and hypoxanthine, aminopterin, and thymidine (HAT), and seeded on a 96-well cell culture plate. The cells were cultured for about 10 days to allow only the hybridomas to grow.

[0077] The pepsin-solubilized type IV collagen fragment and the type IV collagen 7S fragment prepared in (1) above were used as antigens, and each was diluted to 1 μg / mL with PBS and dispensed into a 96-well plate (Nunc Maxisorp, 469914) at 100 μL / well. After adsorbing each antigen overnight at 4°C, the plate was blocked with blocking buffer (1% BSA, 3% sucrose, 0.1% ProClin300 in PBS) for 2 hours. The culture supernatant of the hybridoma prepared above was then added and incubated for 1 hour. After washing four times with washing buffer (0.05% Tween 20 in PBS), 100 μL / well of 5000-fold diluted POD-Goat anti-mouse IgGFcγ (Jackson Immuno Research, 115-036-071) was added to each well. After 30 min of incubation, each well was washed four times with washing buffer and developed with TMB solution (Nacalai, 05298). 2 SO 4The reaction was stopped at 400 rpm and the absorbance of each well was measured using a plate reader.

[0078] In this way, hybridoma strains (CIV09 strain, CIV13 strain) producing anti-type IV collagen 7S domain monoclonal antibodies (anti-7S domain antibodies) that bind to the plate on which the type IV collagen 7S fragment is immobilized were selected. The CIV09 strain and CIV13 strain selected in this way bind to both the pepsin-solubilized type IV collagen fragment and the type IV collagen 7S fragment. On the other hand, a hybridoma strain (CIV03 strain) producing an anti-type IV collagen monoclonal antibody that binds to the plate on which the pepsin-solubilized type IV collagen fragment is immobilized but does not bind to the plate on which the type IV collagen 7S fragment is immobilized was selected. Each hybridoma strain was cultured in a serum-free medium (Hybridoma SFM, Gibco), and the resulting culture supernatant was purified using a protein A column to obtain antibody solutions containing each anti-type IV collagen 7S domain monoclonal antibody and anti-type IV collagen monoclonal antibody, respectively. The protein concentration of each antibody solution was determined by absorbance at a wavelength of 280 nm.

[0079] (3) Confirmation of reaction specificity of monoclonal antibodies The IV collagen 7S fragment and pepsin-solubilized IV collagen fragment prepared in (1) above were used as antigens, and each was diluted with PBS to 1 μg / mL, and dispensed into a 96-well plate at 100 μL / well. After each antigen was adsorbed overnight at 4° C., the plate was blocked with the blocking buffer for 2 hours. Next, each antibody solution obtained in (2) above was serially diluted from 1000 ng / mL to 3.9 ng / mL, dispensed at 100 μL / well, and incubated for 1 hour. After washing four times with the washing buffer, the POD-Goat anti-mouse IgG Fcγ diluted 5000-fold was added to each well at 100 μL / well. After incubation for 30 minutes, each well was washed four times with the washing buffer, and color development was performed with TMB solution. The color development reaction was monitored by H 2 SO 4 The reaction was stopped at 400 rpm and the absorbance of each well was measured using a plate reader.

[0080] The relationship between the concentration (Ab conc. (ng / mL)) of each monoclonal antibody and the absorbance (ratio of wavelengths 450 nm and 630 nm (A450 / 630), the same below) when the type IV collagen 7S fragment prepared in (1) above was used as an antigen is shown in FIG. 2, and the relationship between the concentration (Ab conc. (ng / mL)) of each monoclonal antibody and the absorbance when the pepsin-solubilized type IV collagen fragment was used as an antigen is shown in FIG. 3. It was confirmed that monoclonal antibodies (CIV09, CIV13: anti-type IV collagen 7S domain monoclonal antibodies) that specifically bind to the type IV collagen 7S fragment obtained by the collagenase treatment and a monoclonal antibody (CIV03: anti-type IV collagen monoclonal antibody) that binds to the pepsin-solubilized type IV collagen fragment but does not bind to the type IV collagen 7S fragment were obtained.

[0081] (4) Preparation of antibody-bound particles Using a carboxyl-amine crosslinker (carbodiimide, Thermo-Fisher Scientific), anti-type IV collagen 7S domain monoclonal antibody CIV09 (hereinafter sometimes referred to as "CIV09 antibody") was chemically bound to magnetic particles (manufactured by Fujirebio Inc.) according to the product manual to obtain CIV09 antibody-immobilized magnetic particles. Similarly, anti-type IV collagen monoclonal antibody CIV03 (hereinafter sometimes referred to as "CIV03 antibody") was chemically bound to magnetic particles to obtain CIV03 antibody-immobilized magnetic particles.

[0082] (5) Preparation of labeled antibodies Anti-type IV collagen 7S domain monoclonal antibody CIV13 (hereinafter sometimes referred to as "CIV13 antibody") was bound to alkaline phosphatase derived from bovine small intestine (manufactured by Oriental Yeast Co., Ltd.) by the method of Yoshitake et al. (Yoshitake et al., J. Biochem. 1982, 92(5), p.1413-1424) to prepare alkaline phosphatase-labeled CIV13 antibody. That is, as is usually done, first, desalted CIV13 antibody was mixed with pepsin in 0.1 M citrate buffer (pH 3.5) and left to stand at 37°C for 1 hour to perform pepsin digestion. After stopping the reaction, gel filtration purification was performed to obtain CIV13 antibody from which the Fc region had been removed. Next, 2-mercaptoethylamine hydrochloride was added to perform thiolation. This was further desalted to obtain the Fab' fragment of the CIV13 antibody.

[0083] The alkaline phosphatase treated with N-(4-maleimidobutyryloxy)-succinimide (GMBS) was mixed with the Fab' fragment of the CIV13 antibody to perform coupling. The reaction was stopped by adding 2-mercaptoethylamine hydrochloride and iodoacetamide to the coupling solution to obtain an alkaline phosphatase-labeled CIV13 antibody (ALP-labeled CIV13 antibody).

[0084] (6) Salt concentration First, 30 μL of the sample was added to 50 μL of particle suspensions (50 mM Tris, 1% BSA, 0.15M to 1.5M NaCl) containing 0.03% of the CIV09 antibody-immobilized magnetic particles prepared in (4) above and different salt concentrations, and reacted for 8 minutes at 37° C. The samples used were serum (patient specimens) from patients with cirrhosis or nonalcoholic steatohepatitis (liver disease), or hepatic cancer, serum (healthy specimens) from healthy subjects, and the type IV collagen 7S fragment (Type IV COL 7S), pepsin-solubilized type IV collagen fragment (Type IV COL), and collagenase partially digested type IV collagen fragment (partially digested Type IV COL) prepared in (1) above, each diluted with Tris buffer (50 mM Tris, 2% BSA, 0.15M NaCl, pH 7.2) to 10 ng / mL. When the particle suspension contains 0.15M, 0.3M, 0.6M, 1.0M, 1.2M, and 1.5M NaCl, the salt concentrations of the reaction systems to which each sample is added are 0.15M, 0.24M, 0.43M, 0.68M, 0.80M, and 0.99M, respectively.

[0085] After the reaction, the cells were separated into B / F by a magnet, washed with Lumipulse (registered trademark) washing solution (manufactured by Fujirebio Inc.), and then immersed in a labeled body solution (50 mM Bis-Tris, 600 mM NaCl, 2% BSA, 1 mM MgCl 2 , 0.3 mM ZnCl 2 ) was added to the mixture and reacted at 37°C for 8 minutes. After B / F separation with a magnet and washing with Lumipulse washing solution, 200μL of Lumipulse (registered trademark) substrate solution (manufactured by Fujirebio) containing 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane·disodium salt (AMPPD) was added, and the enzyme reaction was carried out at 37°C for 4 minutes, and the amount of luminescence at a wavelength of 463 nm was measured. Similarly, a standard solution (50mM Tris, 0.15M NaCl, 2% BSA, pH7.2) containing the type IV collagen 7S fragment of known concentration was measured to create a calibration curve. Using the created calibration curve, the concentration of the human type IV collagen fragment containing the 7S domain in each sample was calculated from the amount of luminescence in each sample.

[0086] The results are shown in Tables 1 to 3 below. In Tables 1 to 3, the salt concentration indicates the salt concentration of the particle suspension, and each value indicates the ratio (%) of the luminescence amount under each condition to the luminescence amount when the salt concentration of the particle suspension is 0.15 M. In addition, in Table 1, each value of the patient samples indicates the average value of samples derived from 15 patients with liver disease (liver disease average), and each value of the healthy subject samples indicates the average value of samples derived from 130 healthy subjects (healthy subject average). Meanwhile, in Table 3, the patient samples and healthy subject samples respectively indicate the values ​​of five of each sample (samples 1 to 5).

[0087] [Table 1]

[0088] [Table 2]

[0089] [Table 3]

[0090] As shown in Tables 1 and 3, when the sample was a type IV collagen 7S fragment (Type IV COL 7S), no change in luminescence was observed depending on the salt concentration. On the other hand, as shown in Table 3, when the salt concentration was 0.3M (0.24M as the salt concentration in the reaction system), the luminescence decreased in both the patient specimen and the healthy subject specimen, and was particularly decreased in the healthy subject specimen. When the salt concentration was further increased to 0.6M-1.0M (0.43M-0.68M as the salt concentration in the reaction system), the luminescence increased in the patient specimen, but the luminescence decreased in the healthy subject specimen as the salt concentration increased, and this tendency was significantly greater than that of the patient specimen. And, as shown in Table 1, when the salt concentration was 0.6M-1.5M (0.43M-0.99M as the salt concentration in the reaction system), the luminescence in the healthy subject specimen was significantly lower. This indicates that non-specific reactions are reduced, especially in the healthy subject specimen, at high salt concentrations.

[0091] Furthermore, as shown in Tables 1 and 2, in contrast to the type IV collagen 7S fragment, for which no change in luminescence intensity was observed depending on the salt concentration, in the case of the pepsin-solubilized type IV collagen fragment (Type IV COL) and the collagenase-partially digested type IV collagen fragment (partially digested Type IV COL) samples, when the salt concentration was increased to 0.6 M or more (0.43 M as the salt concentration in the reaction system), the luminescence intensity increased with an increase in the salt concentration. This is considered to be because the 7S domain is masked in fragments containing a part or all of the TH domain other than the type IV collagen 7S fragment contained in the pepsin-solubilized type IV collagen fragment and the collagenase-partially digested type IV collagen fragment, and the masking was removed by increasing the salt concentration.

[0092] From these findings, it is considered that the samples contain a wide variety of human type IV collagen fragments containing the 7S domain, including collagen fragments in which the 7S domain is masked and less exposed, such as fragments containing the TH domain contained in the pepsin-solubilized type IV collagen fragments and the collagenase-partially digested type IV collagen fragments, but that by increasing the salt concentration, the degree of exposure becomes constant, allowing more accurate measurement values ​​to be obtained.

[0093] From the above, it was confirmed that non-specific reactions in healthy human samples are particularly reduced in the sandwich immunoassay of the present invention, which uses a combination of anti-type IV collagen 7S domain monoclonal antibody-immobilized magnetic particles and labeled anti-type IV collagen 7S domain monoclonal antibody, by setting the salt concentration of the reaction system to 0.35 M or more (e.g., 0.43 M (salt concentration of particle suspension 0.6 M) or more).

[0094] (7) Consideration of the sensitivity of the measurement system As a sample, the IV collagen 7S fragment prepared in (1) above was diluted by weight with a standard solution diluent (50 mM Tris, 0.15 M NaCl, 2% BSA, pH 7.2) to prepare 8 types of dilution series of standard solutions. The amount of luminescence was measured in the same manner as in (6) above, except that the salt concentration of the particle suspension was set to 0.6 M and the prepared samples were used, and the concentration of the human IV collagen fragment containing the 7S domain in each sample was calculated using a calibration curve. Each low concentration standard solution (2.0 ng / mL or less) was measured 20 times, and the others were measured in duplicate.

[0095] FIG. 4 shows the relationship between the concentration (7S conc. (ng / mL)) of IV collagen 7S fragment in the standard solution and the amount of luminescence (Signal, average value). FIG. 5 shows the relationship between the actual concentration (7S conc. (ng / mL)) of IV collagen 7S fragment in the standard solution and the coefficient of variation (Value CV (%), average value) of the concentration (quantitative value) of human IV collagen fragment containing the 7S domain quantified by the calibration curve. The detection limit was the concentration at which the average value + 2SD of the measured count at 0 ng / mL was lower than the average value - 2SD (SD: standard deviation) value of the amount of luminescence (Signal) of the low concentration standard solution. The quantification limit was the concentration at which the coefficient of variation (Value CV (%)) was 10% or less. From FIG. 4, the detection limit was 0.1 ng / mL or less, and from FIG. 5, the quantification limit was 0.3 ng / mL or less.

[0096] <Comparison with conventional method (comparison example)> Example 2 As samples, sera from 30 healthy subjects (healthy subjects group), sera from 56 patients with cirrhosis (cirrhosis group), and sera from 38 patients with NASH (NASH group) were used. Except for using these samples, the luminescence amount was measured in the same manner as in (7) of Example 1 above, and the concentration of the fragment containing the type IV collagen 7S domain in each sample was calculated using the calibration curve.

[0097] Comparative Example 1 The same samples as in Example 2 were subjected to measurement according to the attached instructions using a type IV collagen 7S kit (manufactured by DENIS Pharma, hereinafter sometimes referred to as "RIA kit"), which is a radioimmunoassay using anti-human type IV collagen rabbit polyclonal antibody, and the concentration of the fragment containing the type IV collagen 7S domain in each sample was calculated. According to the RIA kit, the fragment containing the type IV collagen 7S domain in the sample ("type IV collagen 7S" in the RIA kit, target substance) reacts with the anti-human type IV collagen rabbit polyclonal antibody to form a fragment containing the type IV collagen 7S domain-anti-human type IV collagen rabbit polyclonal antibody complex (complex 1). Next, the iodized fragment containing the type IV collagen 7S domain ("type IV collagen 7S ( 125 When anti-human type IV collagen rabbit polyclonal antibody (labeled antigen) is added, the anti-human type IV collagen rabbit polyclonal antibody that has not been able to bind to the target substance in the specimen reacts with the labeled antigen to form a labeled antigen-anti-human type IV collagen rabbit polyclonal antibody complex (complex 2). When anti-rabbit γ-globulin goat serum (goat antibody) is added to this, the goat antibody reacts with complex 2 to form a labeled antigen-anti-human type IV collagen rabbit polyclonal antibody-goat antibody complex, which precipitates. This allows the concentration of the target substance (fragment containing the type IV collagen 7S domain) to be determined by measuring the radioactivity of the precipitate after removing the unreacted labeled antigen.

[0098] (correlation) FIG. 6 shows the correlation between the measurement results of Example 2 (CL 7S: concentration (ng / mL) of a fragment containing a type IV collagen 7S domain in a sample) and the measurement results of Comparative Example 1 (RIA 7S: concentration (ng / mL) of a fragment containing a type IV collagen 7S domain in a sample). FIG. 7 shows the correlation for only the healthy subject group. FIG. 6 does not include LC51 and LC28 (i.e., liver cirrhosis group: 54 cases). As shown in FIG. 6, the correlation between the measurement results (CL 7S) by the sandwich immunoassay combining the anti-type IV collagen 7S domain monoclonal antibody immobilized magnetic particles of the present invention and the labeled anti-type IV collagen 7S domain monoclonal antibody and the measurement results (RIA 7S) by the RIA kit was very high, with a correlation coefficient of 0.947. However, as shown in FIG. 7, the correlation coefficient was low at 0.581 for the healthy subject group alone, and it was found that the measurement values ​​of healthy subject samples were clearly reduced by the sandwich immunoassay of the present invention. In other words, the sandwich immunoassay using the monoclonal antibody of the present invention does not reduce the measurement values ​​in the cirrhosis group and NASH group, but can reduce only the measurement values ​​in the healthy group, indicating that it is possible to distinguish between patient groups and healthy groups with higher accuracy than conventional RIA kits.

[0099] (Diagnostic evaluation 1) Example 3 As samples, serum samples from 25 healthy subjects (healthy subject group 1) and serum samples from 30 healthy subjects (healthy subject group 1) were used. Except for using these samples, the luminescence amount was measured in the same manner as in Example 2 above, and the concentration of the fragment containing the type IV collagen 7S domain in each sample was calculated using the calibration curve.

[0100] Comparative Example 2 The concentration of the type IV collagen 7S domain-containing fragment in each sample was calculated using an RIA kit in the same manner as in Comparative Example 1, except that the same samples as in Example 3 were used.

[0101] 〔distribution〕 For healthy subject group 1, the distribution of the measurement results (measured value: concentration (ng / mL) of the fragment containing the type IV collagen 7S domain in the sample) of Example 3 ((b) CL 7S) and Comparative Example 2 ((a) RIA 7S) is shown in Figure 8. For healthy subject group 2, the distribution of the measurement results of Example 3 ((b) CL 7S) and Comparative Example 2 ((a) RIA 7S) is shown in Figure 9.

[0102] As shown in Figures 8 and 9, in the sandwich immunoassay using the monoclonal antibody of the present invention, the measurement values ​​were clearly lower in both healthy subject groups compared to the RIA kit, confirming that the false positive rate in the healthy subject groups could be reduced.

[0103] (Diagnostic evaluation 2) The diagnostic accuracy of Example 2 (CL 7S) and Comparative Example 1 (RIA 7S) was compared by ROC (Receiver Operating Characteristic) analysis. The ROC curve (FPR (false positive rate, 1-specificity)-TPR (positive rate, sensitivity) curve) of the cirrhosis group is shown in FIG. 10, and the ROC curve of the NASH group is shown in FIG. 11. As shown in FIG. 10, in the cirrhosis group, the AUC value in Comparative Example 1 (RIA 7S) was 0.824, whereas the AUC value in Example 2 (CL 7S) was 0.898, indicating that the diagnostic accuracy of cirrhosis is particularly high in the sandwich immunoassay using the monoclonal antibody of the present invention. Also, as shown in FIG. 11, in the NASH group, the AUC value in Comparative Example 1 (RIA 7S) was 0.698, whereas the AUC value in Example 2 (CL 7S) was 0.846, indicating that the diagnostic accuracy of NASH is particularly high in the sandwich immunoassay of the present invention.

[0104] (Measurable aspects of type IV collagen) In the correlation between Example 2 and Comparative Example 1, among the specimens from the cirrhosis group, specimens (LC51, LC28) were found in which the measured values ​​in Comparative Example 1 were significantly lower than those in Example 2. Therefore, the forms of type IV collagen contained in the specimen from the cirrhosis group with this large deviation (LC51), the specimen from the cirrhosis group with a relatively small deviation in the measured values ​​(LC58), and the specimen from the healthy subject group (healthy subject specimen) were compared.

[0105] Example 4 First, 500 μL of each sample was diluted with fractionation buffer (50 mM Tris, 150 mM NaCl, 0.05% CHAPS, 0.09% NaN 3 The solution was applied to a Superose 6 FPLC 10 / 300 equilibrated with 100% ethanol (pH 7.2), developed at 0.5 mL / min of the fractionation buffer, and gel filtered. Fractions were divided into 0.25 mL samples, and the luminescence (CL signal) of each fraction was measured in the same manner as in (7) of Example 1 above. As a control sample, the type IV collagen 7S fragment prepared in (1) above was also fractionated in the same manner and measured.

[0106] Comparative Example 3 For comparison, the luminescence intensity of each fraction of each sample was measured in the same manner as in Example 4, except that CIV03 antibody-immobilized magnetic particles were used as the antibody-immobilized magnetic particles.

[0107] 〔result〕 The luminescence intensity (solid line) of each fraction of specimen LC58 when CIV09 antibody-immobilized magnetic particles were used (Example 4) is shown in FIG. 12(a), the luminescence intensity (solid line) of each fraction of specimen LC51 is shown in FIG. 12(b), and the luminescence intensity (solid line) of each fraction of a specimen from a healthy subject is shown in FIG. 12(c). In FIG. 12(a)-(b), the luminescence intensity of each fraction of type IV collagen 7S fragment (Type IV COL 7S, dotted line) is also shown. In addition, the luminescence intensity of each fraction of specimen LC58 when CIV09 antibody-immobilized magnetic particles were used (Example 4, solid line) or when CIV03 antibody-immobilized magnetic particles were used (Comparative Example 3, dotted line) is shown in FIG. 13(a), and the luminescence intensity of each fraction of type IV collagen 7S fragment is shown in FIG. 13(b).

[0108] As shown in FIG. 12(a), in specimen LC58, a signal was detected only in the high molecular weight fraction. On the other hand, as shown in FIG. 12(b), in specimen LC51, which showed a large deviation between Example 2 and Comparative Example 1, a signal was detected not only in the high molecular weight fraction but also in the fraction with a molecular weight corresponding to the type IV collagen 7S fragment obtained by collagenase treatment. Furthermore, as shown in FIG. 13(a) to (b), when the measurement was performed using CIV03 antibody-immobilized magnetic particles, no signal was detected in any fraction of the type IV collagen 7S fragment (FIG. 13(b)), and similarly, no signal was detected in the fraction corresponding to the type IV collagen 7S fragment in specimen LC58 (FIG. 13(a)).

[0109] That is, in order to comprehensively measure all "fragments containing the 7S domain of type IV collagen" in samples derived from patients with cirrhosis, etc., including the miniaturized 7S fragments of type IV collagen, a sandwich immunoassay using at least a monoclonal antibody (e.g., CIV03 antibody: Comparative Example 3) that binds to pepsin-solubilized 7S fragments of type IV collagen but does not bind to the 7S fragments of type IV collagen as a capture antibody or a labeling antibody is not sufficient. Therefore, it was shown that the sandwich immunoassay of the present invention, in which a monoclonal antibody specific to the 7S domain of type IV collagen is used as both the capture antibody and the labeling antibody, can more accurately measure fragments containing the 7S domain of type IV collagen in samples derived from patients with cirrhosis, etc.

[0110] (Quantitative evaluation) Dilution linearity was examined for samples in which the measured values ​​were different between Example 2 and Comparative Example 1. When the measured values ​​were obtained by a specific reaction, dilution linearity was observed because low measured values ​​were obtained according to the dilution rate when the sample was diluted and measured.

[0111] Example 5 As samples, each specimen from the liver cirrhosis group was diluted 5-fold or 10-fold with a standard diluent (50 mM Tris, 0.15 M NaCl, 2% BSA, pH 7.2) and used. Except for using these specimens, the luminescence amount was measured in the same manner as in Example 2 above, and the concentration of the fragment containing the type IV collagen 7S domain in each specimen was calculated using the calibration curve.

[0112] Comparative Example 4 The concentration of the type IV collagen 7S domain-containing fragment in each sample was calculated using an RIA kit in the same manner as in Comparative Example 1, except that the same samples as in Example 5 were used.

[0113] 〔result〕 Among the samples from the cirrhosis group, for LC27, LC28, LC37, LC51, and LC58, including LC51 and LC28, which showed a large discrepancy between Example 2 and Comparative Example 1 above, the measurement values ​​and their recovery rates (measurement value x dilution ratio / measurement value at dilution ratio 1 x 100 (%)) in Example 5 (CL 7S) and Comparative Example 4 (RIA 7S) are shown in Table 4 below.

[0114] [Table 4]

[0115] As shown in Table 4, in all samples, the sandwich immunoassay using the monoclonal antibody of the present invention achieved a recovery rate of nearly 100% and confirmed dilution linearity (quantitativeness), whereas the RIA kit did not provide dilution linearity.

[0116] (Analysis of samples with discrepant measurements) Example 6 The specimen LC51, which showed the largest deviation in the measured values ​​between Example 2 and Comparative Example 1, was subjected to gel filtration in the same manner as described above (Measurable form of type IV collagen), and the luminescence (signal) of each fraction was measured. In addition, the luminescence of each fraction was measured in the same manner, except that CIV03 antibody-immobilized magnetic particles were used as the control antibody-immobilized magnetic particles. Furthermore, each of the obtained fractions was divided into pools 1 to 6, and the luminescence (signal) of each pool was measured in the same manner as in Example 1 (7). On the other hand, the specimen LC51 before gel filtration was also measured in the same manner, and the measured value was taken as a direct measurement value.

[0117] Comparative Example 5 The luminescence intensity of each pool was measured using an RIA kit in the same manner as in Comparative Example 1, except that the same pool as in Example 6 was used. On the other hand, the LC51 of the sample before gel filtration was also measured in the same manner and was taken as a direct measurement value.

[0118] 〔result〕 For specimen LC51, the luminescence intensity of each fraction when CIV09 antibody-immobilized magnetic particles were used (Example 6, solid line) and when CIV03 antibody-immobilized magnetic particles were used (dotted line) are shown in Figure 14. In addition, the concentration of the fragment containing the type IV collagen 7S domain in each pool was calculated as the pool recovery value (ng / mL) from the measured value of each pool. For specimen LC51, the pool recovery value of each pool when a sandwich immunoassay using a monoclonal antibody (Example 6, CL 7S, solid line) and an RIA kit were used (Comparative Example 5, RIA 7S, dotted line) are shown in Figure 15.

[0119] In addition, the total of the pool recovery values ​​of pools 1 to 6 was used as the recovery value, and the recovery rate was calculated from each direct measurement value using the following formula: recovery rate (%) = recovery value / direct measurement value × 100. The recovery rate in the sandwich immunoassay using the monoclonal antibody of the present invention (Example 6, CL 7S) was 87%, whereas the direct measurement value in the RIA kit (Comparative Example 5, RIA) was low, with a recovery rate of 237%, which was significantly higher than 100%. From this, it is presumed that the anti-human type IV collagen polyclonal antibody used in the CIV03 antibody and the RIA kit is greatly influenced by substances present in the sample and sites other than the 7S domain of type IV collagen, and it is considered that the measurement using these antibodies did not give a correct value. On the other hand, it was shown that the sandwich immunoassay using the monoclonal antibody of the present invention was able to specifically detect a fragment containing the 7S domain of type IV collagen. [Industrial Applicability]

[0120] According to the present invention, it is possible to provide a measurement method that can reduce non-specificity for samples derived from healthy individuals (negative samples) without reducing specificity for samples derived from patients (positive samples), and that can measure fragments containing the human type IV collagen 7S domain in a sample with high accuracy, and a kit for use therein.

[0121] Fragments containing the 7S domain of human type IV collagen in the blood are primarily used as biomarkers for diagnosing liver cirrhosis and nonalcoholic steatohepatitis (NASH). By using the present invention to aid in diagnosis, it is possible to improve diagnostic accuracy and sufficiently reduce nonspecificity for samples derived from healthy individuals, thereby enabling screening and differentiation of patients in the early stages of liver fibrosis.

Claims

1. The method comprises measuring a fragment containing the human type IV collagen 7S domain in a sample by sandwich immunoassay using a capture antibody in which a first monoclonal antibody capable of specifically binding to the human type IV collagen 7S domain is immobilized on a carrier, and a labeled antibody in which a second monoclonal antibody capable of specifically binding to the human type IV collagen 7S domain is bound to a labeling substance, a salt concentration in a reaction system between the fragment containing the 7S domain of human type IV collagen and the first monoclonal antibody is 0.35 to 0.99 M; Measurement method.

2. a capture step of contacting the sample with the capture antibody and capturing the fragment containing the human type IV collagen 7S domain with the capture antibody; a labeling step of contacting the labeled antibody with the fragment containing the human type IV collagen 7S domain captured by the capture antibody after the capture step, thereby labeling the fragment containing the human type IV collagen 7S domain captured by the capture antibody; The method of claim 1 , comprising:

3. A kit for use in the measurement method according to claim 1 or 2, A first monoclonal antibody capable of specifically binding to the 7S domain of human type IV collagen, and a carrier capable of binding to the first monoclonal antibody; a second monoclonal antibody capable of specifically binding to the 7S domain of human type IV collagen; and a labeling substance capable of binding to the second monoclonal antibody; Including the kit.

4. A capture antibody in which a first monoclonal antibody is immobilized on the carrier; a labeled antibody in which a second monoclonal antibody is bound to the label; The kit of claim 3 , comprising:

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