Method for determining a fragment containing the 7S domain of human type IV collagen and a kit for use in the method

Abstract

The present invention provides a measurement method, which uses a capture antibody formed by fixing a first monoclonal antibody capable of specifically binding to the human type IV collagen 7S domain on a carrier and a labeled antibody formed by binding a second monoclonal antibody capable of specifically binding to the human type IV collagen 7S domain to a labeling substance, to measure a fragment containing the human type IV collagen 7S domain in a sample by sandwich immunoassay.

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 the measuring method. Background Art

[0002] Type IV collagen is the main protein that constitutes the basement membrane together with laminin, and has a trimer with a helical structure composed of three α chains as a structural unit. Although the subunits that constitute the trimer are different depending on the tissue, in many tissues such as the liver, the trimer is composed of two α1 chains and one α2 chain. Type IV collagen molecules have structures unique to type IV collagen molecules at both ends of the TH domain (Triple Helical collagenous domain) that forms the helical structure. The structure at the N terminal is called the 7S domain, and the structure at the C terminal is called the NC1 domain (Noncollagenous domain). The NC1 domain at the C terminal forms a dimer with the NC1 domain of other type IV collagen molecules, and the 7S domain at the N terminal forms a tetramer with the 7S domain of other type IV collagen molecules. These polymers are firmly bound by covalent bonds, and through the polymerization of these, type IV collagen molecules form a mesh structure (Chicken-wire network).

[0003] As is known to all, in the human liver, liver fibrosis gradually progresses and develops into cirrhosis or liver cancer due to chronic viral hepatitis of type B or type C or chronic alcoholic hepatitis, progressive non-alcoholic steatohepatitis (NASH), etc., and at this time, type IV collagen increases. Since the amount of type IV collagen leaking from the basement membrane into the blood increases with the progress of such liver fibrosis, the blood concentration of components derived from type IV collagen molecules becomes an indicator of the progress of liver fibrosis. In particular, it is believed that the 7S domain, which is firmly bound by a covalent bond, is not easily affected by proteolytic enzymes and is also stable in the blood, and is therefore used as a biomarker for the diagnosis of the degree of progression (severity) of liver fibrosis or liver cirrhosis.

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

[0005] In addition, the following method is described in Japanese Patent Gazette No. 2-1553 (Patent Document 1): a monoclonal antibody that cross-reacts with the pepsin-solubilized human type IV collagen 7S domain is used as an enzyme-labeled antibody, a monoclonal antibody that only reacts with the human type IV collagen 7S domain is bound to a solid phase support as an antibody-bound solid phase support (capture antibody), and the human type IV collagen 7S domain is quantified by a one-step sandwich immunoassay.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application Laid-Open No. 2-1553 Summary of the invention

[0009] Technical problem to be solved by the invention

[0010] However, in the type IV collagen 7S kit, a polyclonal antibody against human type IV collagen is used, and since the principle is based on competitive inhibition of binding, the measurement accuracy is still insufficient. In particular, the inventors have found the following technical problems: the measurement value of samples from healthy people becomes high, the false positive rate becomes high, and it is not suitable for screening or identifying patients in the early stage of liver fibrosis.

[0011] In addition, in the blood from patients with liver fibrosis (cirrhosis patients, NASH patients, etc.), there are collagen fragments of various forms, from small to large molecules, as type IV collagen fragments containing the 7S domain. The present inventors have found that the type IV collagen·7S kit or the method described in patent document 1 using a monoclonal antibody that cross-reacts with the pepsin-solubilized human type IV collagen 7S domain as an enzyme-labeled antibody cannot fully detect small-molecule collagen fragments, and therefore it is difficult to cover all of these collagen fragments for measurement. As a result, there is a technical problem that the measurement accuracy sometimes decreases depending on the sample.

[0012] Furthermore, the present inventors have found that these conventional methods are also prone to be affected by sites other than the 7S domain of type IV collagen or other components contained in the sample, and thus have a technical problem in that the measurement accuracy is easily reduced.

[0013] The present invention has been completed in view of the technical problems newly discovered by the above-mentioned inventors, and its purpose is to provide a measurement method and a kit therefor, which will not reduce the specificity for samples derived from patients (positive samples), can reduce the non-specificity for samples derived from healthy people (negative samples), and can measure the fragment containing the human type IV collagen 7S domain in the sample with high precision.

[0014] Technical solutions for solving technical problems

[0015] In order to solve the above technical problems, the present inventors have conducted intensive research, and as a result, they have found that: by combining a sandwich immunoassay with a monoclonal antibody that can perform site-specific binding to the 7S domain of human type IV collagen, it will not reduce the measurement value of the sample (positive sample) derived from the patient, and can fully reduce the measurement value of the sample (negative sample) derived from a healthy person, and in particular, in the low concentration area of ​​the fragment containing the 7S domain of human type IV collagen, it is possible to achieve a higher measurement accuracy than before. In addition, according to this method, as a type IV collagen fragment containing the 7S domain, any form of collagen fragment can be comprehensively measured, and further, it is not easily affected by substances present in the sample or sites other than the 7S domain of type IV collagen, thereby achieving a higher measurement accuracy than before, and completing the present invention.

[0016] The aspects of the present invention obtained from this finding are as follows. [1]

[0018] A measurement method uses a capture antibody formed by immobilizing a first monoclonal antibody capable of specifically binding to the human type IV collagen 7S domain on a carrier and a labeled antibody formed by binding a second monoclonal antibody capable of specifically binding to the human type IV collagen 7S domain to a labeling substance, to measure a fragment containing the human type IV collagen 7S domain in a sample by sandwich immunoassay. [2]

[0020] The measuring method according to [1], comprising:

[0021] a capturing step of contacting the sample with the capture antibody to capture the fragment containing the human type IV collagen 7S domain with the capture antibody; and

[0022] A labeling step, after the capturing step, wherein the labeled antibody is brought into contact with the fragment containing the human type IV collagen 7S domain captured by the capturing antibody to label the fragment containing the human type IV collagen 7S domain captured by the capturing antibody. [3]

[0024] The measurement method according to [1] or [2], wherein the salt concentration of the reaction system of the fragment comprising the human type IV collagen 7S domain and the first monoclonal antibody is 0.35 M or more. [4]

[0026] A kit for use in the assay method described in any one of [1] to [3], the kit comprising:

[0027] A first monoclonal antibody capable of specifically binding to the 7S domain of human type IV collagen; a carrier capable of binding to the first monoclonal antibody;

[0028] 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. [5]

[0030] The kit according to [4], comprising:

[0031] A capture antibody formed by immobilizing the first monoclonal antibody on the carrier; and

[0032] The second monoclonal antibody binds to the labeling substance to form a labeled antibody.

[0033] Effects of the Invention

[0034] According to the present invention, a measurement method and a kit therefor can be provided, which will not reduce the specificity for samples derived from patients (positive samples), can reduce the nonspecificity for samples derived from healthy persons (negative samples), and can measure fragments containing the human type IV collagen 7S domain in the sample with high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a photograph of electrophoresis showing the results of SDS-PAGE in the collagenase treatment of Example 1(1).

[0036] Figure 2 This is a graph showing the relationship between the concentration of each monoclonal antibody and the absorbance when the type IV collagen 7S fragment is used as an antigen.

[0037] Figure 3 This is a graph showing the relationship between the concentration of each monoclonal antibody and the absorbance when a pepsin-solubilized type IV collagen fragment is used as an antigen.

[0038] Figure 4 This is a graph showing the relationship between the concentration of the type IV collagen 7S fragment in the standard solution and the luminescence intensity.

[0039] Figure 5 This is a graph showing the relationship between the actual concentration of the type IV collagen 7S fragment in the standard solution and the coefficient of variation of the quantitative value.

[0040] Figure 6 It is a dispersion diagram showing the correlation between the measurement result of Example 2 (CL 7S) and the measurement result of Comparative Example 1 (RIA7S).

[0041] Figure 7It 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) only for the healthy subject group.

[0042] Figure 8 This is a graph showing the distribution of the measured values ​​of the healthy subject group 1, Example 3 ((b) CL 7S) and Comparative Example 2 ((a) RIA 7S).

[0043] Fig. 9 This is a graph showing the distribution of the measured values ​​of the healthy subject group 2, Example 3 ((b) CL 7S) and Comparative Example 2 ((a) RIA 7S).

[0044] Fig.10 It is a graph showing the ROC curves of Example 2 (CL 7S) and Comparative Example 1 (RIA 7S) for the liver cirrhosis group.

[0045] Fig.11 It is a graph showing the ROC curves of Example 2 (CL 7S) and Comparative Example 1 (RIA 7S) for the NASH group.

[0046] Fig.12 The graphs show the luminescence intensity of each fraction of sample LC58 (a), the luminescence intensity of each fraction of sample LC51 (b), and the luminescence intensity of each fraction of a healthy subject sample (c) when using CIV09 antibody-immobilized magnetic particles.

[0047] Fig.13 The graphs show 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.

[0048] Fig.14 This is a graph showing the luminescence amount of each fraction of the sample LC51 when using CIV09 antibody-immobilized magnetic particles or CIV03 antibody-immobilized magnetic particles.

[0049] Fig.15 Yes means Fig.14 A graph of the quantitative values ​​(pool recovery values) of each of pools 1 to 6. DETAILED DESCRIPTION

[0050] Hereinafter, the present invention will be described in detail based on preferred embodiments of the present invention.

[0051] <Method for determining the fragment containing the 7S domain of human type IV collagen>

[0052] The present invention provides a method for measuring a fragment containing the human type IV collagen 7S domain (hereinafter referred to as "the measuring method of the present invention" depending on the circumstances), which uses a capture antibody formed by fixing a first monoclonal antibody capable of specifically binding to the human type IV collagen 7S domain on a carrier and a labeled antibody formed by binding a second monoclonal antibody capable of specifically binding to the human type IV collagen 7S domain to a labeling substance, to measure the fragment containing the human type IV collagen 7S domain in a sample by sandwich immunoassay.

[0053] In the present invention, as a "sample", there is no particular limitation as long as there is a sample containing a fragment of the human type IV collagen 7S domain. Generally speaking, a blood sample collected from a diagnostic subject (human) is used, and as the blood sample, serum, plasma, whole blood can be cited, preferably serum or plasma, more preferably serum. In addition, as the sample, it can be a sample obtained by appropriately diluting or suspending with the following diluent as needed, or it can be a sample that has been pre-treated. As the pre-treatment, for example, collagenase treatment can be cited.

[0054] (fragment containing the 7S domain of human type IV collagen)

[0055] In the sample (preferably a blood sample), as a component derived from human type IV collagen molecules, there may be fragments of various forms of human type IV collagen molecules ranging from small molecules to large molecules. As forms of such fragments, for example, there may be mentioned: a fragment consisting of a 7S domain without a TH domain and an NC1 domain (hereinafter referred to as a "type IV collagen 7S fragment" according to circumstances); a fragment consisting of a TH domain without an NC1 domain and a 7S domain; a fragment consisting of an NC1 domain without a TH domain and a 7S domain; and a fragment comprising all or part of each of two or more of these domains; and a complex of these fragments with other basement membrane components, such as laminin. It should be noted that, in the present invention, the “7S domain” includes any one of the 7S domains that do not form a multimer and the 7S domains that form a dimer to a tetramer, the “TH domain” includes any one of the TH domains that do not form a helical structure and the TH domains that form the helical structure, and the “NC1 domain” includes any one of the NC1 domains that do not form a multimer and the NC1 domains that form a dimer.

[0056] In the present invention, "IV collagen 7S fragment" preferably refers to a gel filtration fraction of a molecular weight equivalent to a tetramer of human IV collagen 7S domain, which is composed of a tetramer of the 7S domain and is a degradation product obtained by further subjecting a pepsin-solubilized IV collagen fragment that has been solubilized by pepsin digestion to collagenase treatment, 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 (e.g., 300 kDa to 440 kDa), or a fragment of human IV collagen containing a 7S domain having a molecular weight equivalent thereto. The time of the collagenase treatment varies depending on the concentration of the added collagenase, and is preferably 30 minutes to 14 hours, for example, and the treatment temperature is preferably 30 to 37°C. It should be noted that, in the present invention, "pepsin-soluble type IV collagen fragment" refers to a human type IV collagen fragment that is solubilized by pepsin digestion, and represents a mixture of two or more of the following: a fragment consisting of a 7S domain (type IV collagen 7S fragment), a fragment consisting of a TH domain, a fragment consisting of an NC1 domain, and a fragment containing all or part of each of two or more of these domains. The pepsin-soluble type IV collagen fragment can be obtained by an appropriate existing known method. For example, it can be obtained by treating an extract containing human type IV collagen molecules from a human placenta with pepsin, or it can be a commercially available product.

[0057] In the present invention, the "fragment containing the 7S domain of human type IV collagen" to be measured can be any fragment of human type IV collagen containing the 7S domain, for example, the type IV collagen 7S fragment; a fragment consisting of all or part of the 7S domain and the TH domain; a fragment consisting of all or part of the 7S domain and the TH domain and the NC1 domain, which may be a single one or a mixture of two or more. According to the measurement method of the present invention, as the fragment containing the 7S domain of human type IV collagen, the type IV collagen 7S fragment containing a small molecular weight or a fragment containing a large molecular weight and in which the 7S domain may not be fully exposed can be measured, and the fragment containing the 7S domain of human type IV collagen can be measured with higher accuracy than before.

[0058] (monoclonal antibody)

[0059] 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 collectively referred to as "anti-7S domain antibody" depending on the circumstances) are monoclonal antibodies capable of specifically recognizing and binding only to the 7S domain of human type IV collagen. That is, the anti-7S domain antibody does not bind to sites other than the 7S domain of human type IV collagen. The antibody is the anti-7S domain antibody, which can be confirmed, for example, in the following manner: it can specifically recognize and bind to the type IV collagen 7S fragment; or it can bind to molecules contained in the fraction with a molecular weight equivalent to the human type IV collagen 7S domain (preferably a tetramer) in the gel filtration fraction of the sample, and can bind not only to the type IV collagen fragment containing the TH domain or the NC1 domain (for example, a pepsin-solubilized type IV collagen fragment solubilized by pepsin treatment), but also to the type IV collagen 7S fragment.

[0060] It should be noted that the "antibody" of the present invention includes, in addition to intact antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, single-chain antibodies, diabodies, etc.) or low-molecular-weight antibodies obtained by combining antibody variable domains.

[0061] The anti-7S domain antibody involved in the present invention can be produced by appropriately adopting or improving a conventionally known method, for example, by a method for producing monoclonal antibodies by fusing antibody-producing cells and myeloma cells (hybridomas) (representatively, by a method based on Kohler and Milstein (Kohler & Milstein, Nature, 256: 495, 1975)).

[0062] Examples of the antibody-producing cells include spleen cells, lymph node cells, peripheral blood leukocytes, and the like of animals immunized with an immunogen (e.g., mice, rats, hamsters, rabbits, monkeys, goats, sheep, donkeys, camels, alpacas, and chickens). Antibody-producing cells can also be used by allowing an immunogen to act in a culture medium on the above-mentioned cells or lymphocytes that have been previously isolated from an animal that has not been immunized.

[0063] In the present invention, the above-mentioned immunogen can be any fragment containing the human type IV collagen 7S domain without any particular limitation, 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 (equivalent to the "collagenase partially digested type IV collagen fragment" in the embodiment).

[0064] As the myeloma cells, various known cell strains can be used. The antibody-producing cells and the myeloma cells may be derived from different animal species as long as they can be fused, but are preferably derived from the same animal species.

[0065] The hybridoma is obtained, for example, by cell fusion between spleen cells obtained from mice immunized with the immunogen and mouse myeloma cells. From the obtained many hybridomas, a hybridoma that produces a monoclonal antibody that shows high reactivity to the type IV collagen 7S fragment is screened, and further screening is performed therefrom as needed, and the monoclonal antibody produced by the obtained hybridoma is subjected to epitope analysis, and the clones that produce the monoclonal antibody that binds to the type IV collagen 7S fragment are identified, thereby obtaining a hybridoma that produces a monoclonal antibody (anti-7S domain antibody) that can specifically bind to the human type IV collagen 7S domain. The monoclonal antibody can be obtained by culturing the hybridoma or from the ascites of a mammal to which the hybridoma is administered.

[0066] In addition, as an anti-7S domain antibody, for example, if a DNA encoding the antibody can be obtained, it can also be produced by a recombinant DNA method. As the recombinant DNA method, there can be cited a method of producing a recombinant antibody by cloning a DNA encoding the above-mentioned antibody from a hybridoma or the antibody-producing cell, inserting it into an appropriate vector, and introducing it into a host cell (e.g., a mammalian cell line, Escherichia coli, a yeast cell, an insect cell, a plant cell, etc.) (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 the expression of the DNA encoding the above-mentioned antibody, the DNA encoding the heavy chain or light chain can be inserted into an expression vector separately to transform the host cell, or the DNA encoding the heavy chain and light chain can be inserted into a single expression vector to transform the host cell (for example, the method described in International Publication No. 94 / 11523).

[0067] The recombinant antibody can be obtained in a substantially pure and uniform form by culturing the host cell and isolating / purifying it from the cultured host cell or the culture fluid. The separation / purification of the recombinant antibody can use the method used in the purification of the usual polypeptide. If a transgenic animal (e.g., cattle, goats, sheep, pigs, etc.) into which an antibody gene is inserted is produced using transgenic animal production technology, monoclonal antibodies derived from the antibody gene can also be obtained in large quantities from the milk of the transgenic animal.

[0068] In the present invention, the first monoclonal antibody and the second monoclonal antibody may be the same or different. When the sample is a blood sample, in the blood sample, the human type IV collagen 7S domain usually forms dimers to tetramers (preferably tetramers), so even if the first monoclonal antibody and the second monoclonal antibody are the same type of antibodies that recognize / bind to the same site, they can each bind to any one of the type IV collagen 7S domains in the multimer.

[0069] (Capture Antibody)

[0070] In the measuring method of the present invention, as the capture antibody for capturing the fragment containing the human type IV collagen 7S domain, a capture antibody obtained by immobilizing the first monoclonal antibody on a carrier is used.

[0071] In the present invention, as a "carrier", there is no particular restriction as long as it can fix and carry the first monoclonal antibody. As the material of such a carrier, there is no particular restriction as long as it is a material generally used in immunoassays, for example, it can be cited: high molecular polymers (such as polystyrene, (meth) acrylate, polymethyl methacrylate, polyimide, nylon, etc.), gelatin, cellulose, nitrocellulose, glass, latex, silica, metals (such as gold, platinum, iron, cobalt, nickel, etc.), metal compounds (such as iron oxide, cobalt oxide, nickel ferrite, etc.) and composite materials or alloys thereof. In addition, as the carrier, for example, it can be a carrier obtained by surface modification with one or more active groups in carboxyl, epoxy, p-toluenesulfonyl, amino, hydroxyl, isothiocyanate, isocyanate, azido, aldehyde, carbonate, allyl, aminooxy, maleimide, thiol, NHS ester (N-hydroxy ester) groups.

[0072] In addition, in the present invention, the shape of the carrier is not particularly limited, and may be any of a plate, a fiber, a membrane, a particle, etc., but particles are preferred from the viewpoint of easy control of the density of the immobilized first monoclonal antibody and easy uniformity. Furthermore, magnetic particles are preferred from the viewpoint of automation / shortening of time.

[0073] When the carrier is a particle, there is no particular limitation on its size, but the particle size is preferably in the range of 0.01 to 100 μm, more preferably in the range of 0.1 to 10 μm. If the particle size of the particle is less than the lower limit, in the case of magnetic particles, there is a tendency that more magnetism and time are required for magnetism collection, while on the other hand, if it exceeds the upper limit, there is a tendency that the specific surface area of ​​the particle becomes smaller, and the amount of the first monoclonal antibody bound to the particle becomes less.

[0074] 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 Japanese Patent Application Laid-Open No. 3-115862, or commercially available particles such as DYNABEADS (manufactured by Thermo Fisher), Magnosphere (manufactured by JSR Co., Ltd.), and MAGURAPITUD (manufactured by Sanyo Chemical Industries, Ltd.) can be appropriately used. In addition, as the particles, one of them can be used alone, or a combination of two or more can be used.

[0075] As the method for producing the capture antibody involved in the present invention, as the method for immobilizing the first monoclonal antibody on the carrier, a conventionally known method or a method based thereon can be appropriately adopted, and the first monoclonal antibody can be directly immobilized on the surface of the carrier or indirectly immobilized. As a direct binding method, for example, a method of imparting the active group to the carrier, or using a substance having these active groups as the carrier, and binding the first monoclonal antibody through a covalent bond based on the active group can be cited.

[0076] As an indirect fixation method, for example, by fixing a substance that binds to the first monoclonal antibody on the surface of the carrier, the first monoclonal antibody is bound to the substance, thereby the first monoclonal antibody can be indirectly fixed on the surface of the carrier. As a substance that binds to the first monoclonal antibody, there is no particular limitation, and for example, a secondary antibody that can bind to the first monoclonal antibody, protein G, protein A, a linker molecule having the active group, and the like can be cited. In addition, when the first monoclonal antibody is biotinylated or avidinized (or streptavidinized), the carrier can also be avidinized (or streptavidinized) or biotinylated, and the antibody can be fixed by the interaction between biotin and avidin (or streptavidin).

[0077] In addition, as the capture antibody involved in the measurement method of the present invention, the capture antibody formed by directly or indirectly immobilizing the first monoclonal antibody on a carrier can be prepared in advance from the first monoclonal antibody and a carrier capable of binding to the first monoclonal antibody, and then supplied to the sandwich immunoassay. The first monoclonal antibody and the carrier capable of binding to the first monoclonal antibody can be supplied separately to the reaction system of the sandwich immunoassay, and the first monoclonal antibody can be immobilized on the carrier in the reaction system, or the first monoclonal antibody can be immobilized on the carrier after forming a complex between the first monoclonal antibody and a fragment containing the human type IV collagen 7S domain. In this case, the combination of "a first monoclonal antibody and a support capable of binding to the first monoclonal antibody" includes any combination of the first monoclonal antibody and the support described above. However, when these are separately provided to the reaction system, the combination of the first monoclonal antibody described in the method of indirectly immobilizing the first monoclonal antibody on the support and a support to which a substance binding to the first monoclonal antibody is immobilized, and the combination of a biotinylated or avidinylated (or streptavidinylated) first monoclonal antibody and an avidinylated (or streptavidinylated) or biotinylated support are preferred.

[0078] The capture antibody involved in the present invention comprises at least the first monoclonal antibody and the carrier, and in addition thereto, may also comprise the linker, blocking agent, etc. as necessary.

[0079] (labeled antibody)

[0080] In the measuring method of the present invention, as the labeled antibody for labeling the fragment containing the human type IV collagen 7S domain, a labeled antibody in which the second monoclonal antibody is bound to a labeling substance is used.

[0081] In the present invention, the "labeling substance" is not particularly limited as long as it is a substance that can be detected by binding to the second monoclonal antibody, 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 I and other radioactive substances; low molecular weight labeling substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); gold particles; avidin; biotin; latex; dinitrophenol (DNP); digoxin (DIG). Among them, as the labeling substance involved in the present invention, from the perspective of having a tendency to be able to perform simple and stable tests and from the perspective of being able to perform simple, rapid and highly sensitive measurements, enzymes and luminescent substances are preferred, and enzymes are more preferred. As the enzyme, for example, horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, luciferase can be cited, but are not limited to these.

[0082] As a method for producing the labeled antibody involved in the present invention, as a method for binding the labeled substance to the second monoclonal antibody, a conventionally known method or a method based on the method can be appropriately adopted, and the binding can be direct or indirect. As such a binding method, the same method as the method cited as the binding method of the carrier to the first monoclonal antibody in the capture antibody involved in the present invention can be cited.

[0083] In addition, as the labeled antibody involved in the assay method of the present invention, the labeled antibody formed by direct or indirect binding of the labeled substance and the second monoclonal antibody can be prepared in advance from the second monoclonal antibody and the labeled substance capable of binding to the second monoclonal antibody, and then supplied to the sandwich immunoassay. Alternatively, the second monoclonal antibody and the labeled substance capable of binding to the second monoclonal antibody can be supplied separately to the reaction system of the sandwich immunoassay, and the labeled substance and the second monoclonal antibody can be bound in the reaction system, or the labeled substance and the second monoclonal antibody can be bound after forming a complex of the second monoclonal antibody and the fragment containing the 7S domain of human type IV collagen. As a combination of "the second monoclonal antibody and the labeled substance capable of binding to the second monoclonal antibody" in this case, any combination of the second monoclonal antibody and the labeled substance mentioned above can be cited. However, when these are supplied separately to the reaction system, a combination of the second monoclonal antibody and the labeled substance bound to the substance binding to the second monoclonal antibody, and a combination of the biotinylated or avidinylated (or streptavidinylated) second monoclonal antibody and the avidinylated (or streptavidinylated) or biotinylated labeled substance are preferred. Examples of the substance that binds to the second monoclonal antibody include the same substances as exemplified as the substance that binds to the first monoclonal antibody.

[0084] The labeled antibody involved in the present invention comprises at least the second monoclonal antibody and the labeling substance, and in addition thereto, may also comprise the linker, blocking agent, etc. as necessary.

[0085] (Sandwich Immunoassay)

[0086] The measuring method of the present invention measures the fragment containing the human type IV collagen 7S domain in a sample by sandwich immunoassay using the capture antibody and the labeled antibody.

[0087] In the present invention, in the "determination", in addition to obtaining the presence and amount of the target substance in the sample in the form of a signal, it also includes the quantitative or semi-quantitative amount of the target substance. In the present invention, the determination of the target substance is preferably performed by detecting the signal generated by the labeling substance and quantifying it as needed. The "signal" includes radiation generated by color development (color development), reflected light, luminescence, fluorescence, radioisotopes, etc., and in addition to signals that can be confirmed with the naked eye, it also includes signals that can be confirmed by a determination method / device corresponding to the type of signal.

[0088] "Sandwich immunoassay" is a method in which a target substance (a "fragment comprising the 7S domain of human type IV collagen" in the present invention) is captured using a capture antibody (a "capture antibody" in the present invention) fixed (solid-phased) on a solid phase, and a detection antibody (a "labeled antibody" in the present invention) bound to a labeled substance is made to recognize the target substance to form a capture antibody-target substance-labeled antibody complex, which is then washed as needed and then a detection method corresponding to the type of labeled substance is performed.

[0089] In addition, in the measurement method of the present invention, "capturing a target substance (a "fragment comprising the 7S domain of human type IV collagen" in the present invention) with a capture antibody (a "capture antibody" in the present invention) fixed (solid-phased) on a solid phase" includes: after forming a complex between a first antibody (a "first monoclonal antibody" in the present invention) not fixed on a solid phase (a "carrier" in the present invention) and the target substance, or after forming a complex between a first antibody not fixed on a solid phase, the target substance and the labeled antibody (or a second antibody described below), the first antibody that formed the complex is fixed on the solid phase, and the target substance (or the target substance and the labeled antibody) is captured on the solid phase via the first antibody. The combination of the first antibody and the solid phase at this time, including its preferred embodiment, is the same as the combination exemplified in the "first monoclonal antibody and a carrier capable of binding to the first monoclonal antibody" of the capture antibody described above.

[0090] Furthermore, in the determination method of the present invention, "making the detection antibody bound to the labeling substance ("labeling antibody" in the present invention) recognize the target substance" includes: after forming a complex between a second antibody not bound to the labeling substance ("second monoclonal antibody" in the present invention) and the target substance, or after forming a complex between the second antibody not bound to the labeling substance, the target substance and the capture antibody (or the first antibody), making the labeling substance bind to the second antibody that forms the complex, so that the labeling substance recognizes 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 at this time, including its preferred embodiment, is the same as the combination exemplified in the "second monoclonal antibody and the labeling substance capable of binding to the second monoclonal antibody" of the above-mentioned labeling antibody.

[0091] As such sandwich immunoassays, for example, there can be mentioned: the sandwich CLIA method which is a method of the chemiluminescent immunoassay (CLIA method), the sandwich CLEIA method which is a method of the chemiluminescent enzyme immunoassay (CLEIA method), the IRMA method (radiometric immunoassay) which is a method of the radioimmunoassay (RIA method), the sandwich ECLIA method which is a method of the electrochemical immunoassay (ECLIA method), and the sandwich FIA method which is a method of the fluorescent immunoassay (FIA method).

[0092] In the determination method of the present invention, the salt concentration of the reaction system of the fragment comprising the human type IV collagen 7S domain and the first monoclonal antibody (preferably the reaction system in which the fragment comprising the human type IV collagen 7S domain is captured by the capture antibody) is 0.35M (M: mol / L, the same below) or more. The salt concentration is more preferably 0.35 to 1.3M, further preferably 0.42 to 1.0M, and further preferably 0.43 to 0.99M. In the determination method of the present invention, in particular, by making the salt concentration within the range, the nonspecific reaction of samples from healthy subjects can be further reduced, and the determination accuracy can be further improved.

[0093] The present inventors have found that in the determination of a fragment containing the 7S domain of human type IV collagen, by making the salt concentration when it is bound to the first monoclonal antibody (preferably when captured with the capture antibody) higher than the salt concentration in the conventional sandwich immunoassay (e.g., about 0.15 M), it is possible to weaken the binding to the 7S domain of human type IV collagen contained in a sample derived from a healthy person, and to maintain / enhance the binding to the 7S domain contained in a sample derived from a patient with liver fibrosis (a cirrhosis patient, a NASH patient, etc.). This is because, as a fragment containing the 7S domain of human type IV collagen, the composition of the collagen fragment contained in the sample derived from the patient is different from that of the collagen fragment contained in the sample derived from the healthy person, and according to the determination method of the present invention, the nonspecificity to the collagen fragment contained in the sample derived from the healthy person is reduced in a high salt concentration. In addition, the following is also considered to be a reason: in the sample, the exposure degree of the 7S domain varies depending on the length of the TH domain contained or the size of the mesh structure of the collagen, and there are even collagen fragments with large molecular weight. Therefore, at low salt concentrations, the measured value obtained by the anti-7S domain antibody and the actual amount of the fragment containing the human type IV collagen 7S domain in the sample are prone to deviation. In contrast, by increasing the salt concentration, the shielding is released, the 7S domain is exposed, and the deviation can be eliminated.

[0094] The reaction system of the fragment containing the human type IV collagen 7S domain and the first monoclonal antibody (preferably a reaction system captured by the capture antibody) is not particularly limited as long as it is a reaction system containing the sample, the first monoclonal antibody (preferably the capture antibody) and the salt. It is preferably an aqueous solution. In addition, it can also contain: physiological saline, purified water; buffers such as MES, Tris, CFB, MOPS, PIPES, HEPES, tris (hydroxymethyl) methylglycine buffer, N-bicine buffer, glycine buffer; stabilized proteins such as BSA; various surfactants. In addition, when a reverse sandwich method or a one-step method is used, the labeled antibody can also be included. In addition, when a salt is included in these other components, the salt concentration of the reaction system also includes the concentration of the salt.

[0095] In addition, when the sample contains salt, the salt concentration of the reaction system also includes the concentration of the salt. Blood samples (whole blood, serum, plasma) generally contain salts equivalent to 140mM to 150mM in total. For example, when a blood sample (whole blood, serum, plasma, etc.) is used as the sample, as shown in the following example, when 30μL of a serum sample as a sample is added to a 50μL suspension of particles (capture antibody) containing 0.6M to 1.5M salt, the salt concentration of the reaction system in which the fragment containing the 7S domain of human type IV collagen is captured by the capture antibody is 0.43M to 0.99M.

[0096] 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 alkali metal chlorides, alkali metal tartrates, alkali metal nitrates, and alkali metal sulfates. The alkali metal ions are preferably sodium ions and potassium ions. In this case, the salt concentration represents a concentration equivalent to the concentration of the alkali metal ions.

[0097] In such a sandwich immunoassay, the complex (capture antibody-fragment containing human type IV collagen 7S domain (target substance)-labeled antibody) is finally formed by contacting the first monoclonal antibody and the carrier (e.g., the capture antibody) capable of binding to the first monoclonal antibody, the sample, and the second monoclonal antibody and the labeling substance (e.g., the labeling antibody) capable of binding to the second monoclonal antibody. There is no particular limitation on such a contact method, and a conventionally known method or a method based on the method can be appropriately adopted. For example, a method of adding the first monoclonal antibody and the carrier (e.g., the capture antibody), and / or the second monoclonal antibody and the labeling substance (e.g., the labeling antibody) to the sample can be cited; or, for example, a method of adding the sample to the capture antibody solution (e.g., a particle suspension when the carrier is a particle) and / or the labeling antibody solution can be cited. In addition, as the sample, a substance diluted or suspended with a diluent can be appropriately used.

[0098] When the first monoclonal antibody and the carrier (e.g., the capture antibody) capable of binding to the first monoclonal antibody, the sample, and the second monoclonal antibody and the labeling substance (e.g., the labeling antibody) capable of binding to the second monoclonal antibody are contacted, it is preferred that the salt concentration when the first monoclonal antibody (preferably the capture antibody) and the sample are contacted satisfies the above conditions, but there are no particular restrictions. For example, in addition to the solvent of the capture antibody solution (e.g., the particle suspension medium when the carrier is a particle), the solvent of the labeling antibody solution, and the diluent, a reaction buffer may be appropriately added. The solvent of the capture antibody solution, the solvent of the labeling antibody solution, the diluent, and the reaction buffer are not particularly limited, and for example, physiological saline, purified water, buffer (sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tris(hydroxymethyl)methylglycine buffer, N-bicine buffer, glycine buffer), etc., may be independently cited. In addition, they may be independently added with a stabilized protein such as BSA, etc. It should be noted that, when these solutions contain salts, the salt concentration of the reaction system also includes the concentration of the salt. In addition, when the sample contains salts, the salt concentration of the reaction system also includes the concentration of the salt.

[0099] In the formation of the complex, the content of the first monoclonal antibody in the reaction system (the total amount of these in the case of a combination of two or more, the same below) is not particularly limited and can be appropriately adjusted according to the type, concentration, detection method, etc. of the sample. Therefore, there is no particular limitation, but from the viewpoint of efficient capture in a short time, for example, it is preferably 0.000001 to 0.01 w / v%, and more preferably 0.00001 to 0.001 w / v%. It should be noted that the symbol "w / v%" in this specification represents weight / volume percentage (g / 100 mL).

[0100] In addition, in the formation of the complex, the content of the second monoclonal antibody in the reaction system (in the case of a combination of two or more, it is the total amount of these, and the same applies below) is not particularly limited and can be appropriately adjusted according to the type, concentration, detection method, etc. of the sample. Therefore, there is no particular limitation. However, from the perspective of efficient capture in a short time, it is preferably 0.000001 to 0.01 w / v%, and more preferably 0.00001 to 0.001 w / v%.

[0101] Furthermore, the conditions for the contacting are not particularly limited and can be appropriately adjusted independently. For example, the contacting can be carried out at 4 to 45°C, preferably 20 to 37°C, pH 6 to 9, preferably pH 6.5 to 8, for about 30 seconds to 12 hours, preferably 1 minute to 1 hour, but is not limited to these conditions.

[0102] Examples of the sandwich immunoassay include: a two-step forward sandwich method (a method in which carrier-based capture (preferably a reaction between a capture antibody and a fragment containing the human type IV collagen 7S domain in a sample) and labeled substance-based recognition (preferably a reaction between a fragment containing the human type IV collagen 7S domain bound to the capture antibody and a labeled antibody) are performed sequentially), a reverse sandwich method (a method in which a complex is preliminarily generated by labeled substance recognition (preferably a reaction between a labeled antibody and a fragment containing the human type IV collagen 7S domain in a sample) and capture with a carrier (preferably a reaction with a capture antibody)), and a one-step method (a method in which a capture antibody, a fragment containing the human type IV collagen 7S domain in a sample and a labeled antibody are reacted simultaneously in one step), and any of these can be used.

[0103] Among them, as the measuring method of the present invention, from the viewpoint of measuring accuracy, the two-step method is preferred, and the forward sandwich method is more preferred. As a form of the measuring method of the present invention of such a forward sandwich method, for example, a measuring method can be cited, which comprises:

[0104] a capturing step of contacting the sample with the capture antibody to capture the fragment containing the human type IV collagen 7S domain with the capture antibody; and

[0105] A labeling step, after the capturing step, wherein the labeled antibody is brought into contact with the fragment containing the human type IV collagen 7S domain captured by the capturing antibody to label the fragment containing the human type IV collagen 7S domain captured by the capturing antibody.

[0106] [Capture process]

[0107] In the capture step, the capture antibody is brought into contact with the sample, and the capture antibody captures the fragment containing the human type IV collagen 7S domain via the binding of the fragment containing the human type IV collagen 7S domain with the first monoclonal antibody, i.e., a first immune complex of the first monoclonal antibody and the fragment containing the human type IV collagen 7S domain is formed. There are no particular restrictions on the method for bringing the capture antibody into contact with the sample and the conditions at that time, but it is preferred to satisfy the salt concentration condition, and the method and conditions described in the above-mentioned method and conditions for bringing the capture antibody, the sample, and the labeled antibody into contact, respectively, can be applied.

[0108] [Cleaning process]

[0109] As the measuring method of the present invention, it is preferred that after the capture step and before the labeling step described below, a washing step is further included to separate the fragment containing the human type IV collagen 7S domain captured by the capture antibody and other inclusions that are not bound to the capture antibody (not captured) and remove the inclusions. There is no particular limitation on the method for removing the inclusions, and a conventionally known method or a method based on it can be appropriately adopted. For example, a method of removing the liquid phase (supernatant) by centrifugation or magnetic collection after the capture step can be cited. In addition, in the washing step, the injection and removal of the washing liquid can be repeated as needed. As the washing liquid, for example, a neutral (preferably pH 6 to 9) known buffer (sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tris (hydroxymethyl) methylglycine buffer, N-bicine buffer, glycine buffer, etc.) can be cited. In addition, each independently can also be a substance obtained by adding a stabilizing protein such as BSA, a surfactant, etc.

[0110] [Marking process]

[0111] In the labeling step, the labeled antibody is brought into contact with the fragment containing the human type IV collagen 7S domain captured by the capture antibody to label the fragment containing the human type IV collagen 7S domain captured by the capture antibody, i.e., a second immune complex of the second monoclonal antibody and the fragment containing the human type IV collagen 7S domain captured by the capture antibody is formed. There are no particular restrictions on the method for bringing the labeled antibody into contact with the fragment containing the human type IV collagen 7S domain captured by the capture antibody and the conditions at that time, and the method and conditions described in the above-mentioned method and conditions for bringing the capture antibody, the sample, and the labeled antibody into contact with each other can be applied.

[0112] Through the above-mentioned capture step and labeling step, the complex (second immune complex) comprising the capture antibody-fragment comprising the human IV collagen type 7S domain-labeling antibody is formed. After the labeling step, in order to remove the labeled antibody that has not formed the complex, a washing step may also be included as needed. As this washing step, it is the same as the above-mentioned washing step.

[0113] In the determination method of the present invention, the labeling substance of the complex (capture antibody-fragment containing human type IV collagen 7S domain-labeling antibody) is determined by a prescribed method corresponding to the labeling substance. For example, when the labeling substance of the labeled antibody is an enzyme, a chromogenic substrate, a luminescent substrate, a chemiluminescent substrate, etc. corresponding to the enzyme is added to detect the signal (coloration, luminescence, etc.) generated by reacting the enzyme with the substrate. Thus, the presence and amount of the fragment containing human type IV collagen 7S domain in the sample can be detected in the form of a signal. In addition, the quantification of the amount of the fragment containing human type IV collagen 7S domain in the sample is generally carried out by comparing with the measured value of a standard solution containing a fragment containing human type IV collagen 7S domain of each concentration. There is no particular limitation on the fragment containing human type IV collagen 7S domain used in the standard solution, and for example, it can be cited: the type IV collagen 7S fragment, the collagenase partially digested type IV collagen fragment. In this case, for example, by examining where the measured value obtained in the sample is located on a calibration curve prepared from the measured value of the standard solution, the amount of the fragment containing the human type IV collagen 7S domain in the sample can be determined.

[0114] The measuring method of the present invention can be used for diagnosis (evaluation of the occurrence or risk) of diseases related to abnormal blood concentration of type IV collagen and its assistance. Here, as "diseases related to abnormal blood concentration of type IV collagen", for example, diseases in which liver fibrosis progresses, for example, viral chronic hepatitis type B and type C, alcoholic hepatitis, non-alcoholic steatohepatitis (NASH), cirrhosis, liver cancer can be mentioned.

[0115] In addition, the determination method of the present invention can also be used for the diagnosis of the progression (severity) of the liver fibrosis or its assistance or identification, or the screening of patients who may cause liver fibrosis. In particular, according to the determination method of the present invention, the specificity for the sample (positive sample) derived from the patient will not be reduced, the non-specificity for the sample (negative sample) derived from the healthy person can be reduced, and high determination accuracy can be achieved in the low concentration region of the fragment containing the human type IV collagen 7S domain. Therefore, it is very useful as a determination method for screening patients with liver fibrosis progression in the early stage from the healthy group and for identification with healthy people.

[0116] <Kit for the assay method of a fragment containing the 7S domain of human type IV collagen>

[0117] The present invention also provides a kit for a method of measuring a fragment comprising a human type IV collagen 7S domain (hereinafter referred to as "the kit of the present invention" as the case may be), which is a kit for the measuring method of the present invention, the kit comprising:

[0118] A first monoclonal antibody capable of specifically binding to the 7S domain of human type IV collagen; a carrier capable of binding to the first monoclonal antibody;

[0119] 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. As the kit of the present invention, the following method is preferred, comprising:

[0120] A capture antibody formed by immobilizing the first monoclonal antibody on the carrier; and

[0121] The second monoclonal antibody binds to the labeling substance to form a labeled antibody.

[0122] 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 labeling antibody contained in the kit of the present invention are as described in the measurement method of the present invention, including preferred embodiments. In addition, 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 labeling antibody contained in the kit of the present invention may be each independently in a solid (powder) state, or in a liquid state dissolved in a buffer solution or the like.

[0123] As a test kit of the present invention, it can also have the structure that should be possessed in common sandwich analysis such as ELISA, CLEIA, ECLIA, CLIA, FIA, immunochromatography, etc. For example, it can also have at least one selected from the group consisting of the standard solution (each concentration), the control reagent, the diluent, the solvent of the capture antibody solution (particle suspension, etc.), the solvent of the labeled antibody solution, the reaction buffer, the cleaning solution and the dilution box. In addition, for example, when the labeling substance is an enzyme, it can also include a substrate or reaction stop solution required for the detection / quantification of the labeling substance. Furthermore, as needed, it can also include a pretreatment solution for pre-treatment of the sample or the instructions for use of the test kit.

[0124] Example

[0125] The present invention is described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples. It should be noted that in each example and comparative example, the symbol "%" refers to weight / volume percentage (w / v%: g / 100mL) unless otherwise specified.

[0126] <Sandwich immunoassay using anti-type IV collagen 7S domain monoclonal antibody>

[0127] (Example 1)

[0128] (1) Collagenase treatment

[0129] 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, the pepsin-soluble type IV collagen fragment (derived from human placenta, Sigma, C-7521) prepared by pepsin digestion was dissolved in 0.5M acetic acid and dialyzed against 50mM Tris (pH7.2) containing 0.2M NaCl and 2mM CaCl2. Then, collagenase (Collagenase "Amano", Wako, 607-19021) was added thereto and cultured at 37°C for 1 hour to further digest the pepsin-soluble type IV collagen fragment (collagenase treatment). The treated solution after collagenase treatment was centrifuged and the supernatant was further separated by gel filtration. Superdex200pg 16 / 60 was used as a gel filtration column, and 1mM EDTA 3Na and PBS were used as gel filtration buffers. Figure 1 In the figure, the results of SDS-PAGE (2-15% PAG Non-Reduce) of the MW Marker (No.1, molecular weight / 1000), pepsin-solubilized type IV collagen fragment (No.2), the treatment solution after collagenase treatment (No.3) and its precipitate (No.4) and supernatant (No.5), and each fraction (GF Fr.3-7; No.6-10) are shown. The fractions with a molecular weight of 250,000 (or a mass of 250 kDa) or more and distributed around 400,000 (300 kDa to 440 kDa) corresponding to the molecular weight of the tetramer of human type IV collagen 7S domain were concentrated and used as the following type IV collagen 7S fragment.

[0130] In the present embodiment and comparative example, the "IV collagen type 7S fragment" is a human IV collagen type fragment composed of the 7S domain without the TH domain, and can be obtained by treating with collagenase and selecting the gel filtration fraction. In addition, as the immunogen described below, a collagenase partially digested IV collagen type fragment is used. The "collagenase partially digested IV collagen type fragment" is a fragment mainly composed of a substance with a molecular weight of 500k or more, which is prepared by digesting a pepsin-solubilized IV collagen type fragment (manufactured by SIGMA) with collagenase at a treatment temperature of 25°C for 1 hour, and is a mixture mainly composed of the IV collagen type 7S fragment and a human IV collagen type fragment composed of a part of the 7S domain and the TH domain. It should be noted that the "pepsin-solubilized IV collagen type fragment" is a fragment prepared by solubilization by pepsin digestion, and is a mixture of a human IV collagen type fragment composed of a 7S domain, a TH domain or an NC1 domain, and a human IV collagen type fragment containing all or part of two or more of them.

[0131] (2) Preparation of monoclonal antibodies

[0132] Monoclonal antibodies were prepared using the "collagenase partially digested type IV collagen fragment" prepared in (1) as an immunogen. Specifically, for BALB / c mice, 10 μg (1 mg / mL) of collagenase partially digested type IV collagen fragment was mixed with an equal amount of complete Freund's adjuvant, and intraperitoneal immunization was performed twice at an interval of two weeks. Furthermore, incomplete Freund's adjuvant was used for two intraperitoneal immunizations 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. On the third day after the final immunization, the spleen was removed from the mouse, dissociated into single cells, and washed three times with RPMI-1640 medium. The mouse myeloma cell line Sp2 / OAg14 in the logarithmic growth phase was washed three times with RPMI-1640 medium, mixed with the spleen cells, and cell fusion was performed. The obtained fusion cells were centrifuged (200×g, 5 minutes) to remove PEG, and then suspended in RPMI-1640 medium containing 10% fetal bovine serum, hypoxanthine, aminopterin, and thymidine (HAT), seeded in a 96-well cell culture plate, and cultured for about 10 days to allow only hybridomas to proliferate.

[0133] Pepsin-soluble type IV collagen fragment and type IV collagen 7S fragment prepared in (1) above were used as antigens, 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 in blocking buffer (1% BSA, 3% sucrose, 0.1% ProClin300 in PBS) for two hours. Next, the culture supernatant of the hybridoma prepared above was added and cultured for 1 hour. After washing 4 times with washing buffer (0.05% Tween 20 in PBS), 100 μL / well of 5000-fold diluted POD-Goat anti-mouse IgG Fcγ (Jackson Immuno Research, 115-036-071) was added to each well. After incubation for 30 minutes, each well was washed four times with a washing buffer, and color was developed with a TMB solution (Nacalai, 05298). The color development reaction was stopped with H2SO4, and the absorbance of each well was measured with an enzyme-labeled instrument.

[0134] Thus, hybridoma strains (CIV09 strain, CIV13 strain) that produce anti-IV collagen 7S domain monoclonal antibodies (anti-7S domain antibodies) that bind to the plate on which the IV collagen 7S fragment is solid-phased are selected. The CIV09 strain and CIV13 strain selected in this way bind to both the pepsin-solubilized IV collagen fragment and the IV collagen 7S fragment. On the other hand, a hybridoma strain (CIV03 strain) that produces anti-IV collagen monoclonal antibodies that bind to the plate on which the pepsin-solubilized IV collagen fragment is solid-phased is selected, but not to the plate on which the IV collagen 7S fragment is solid-phased is solid-phased is selected. Each hybridoma strain is cultured with a serum-free medium (Hybridoma SFM, Gibco), and purified from the obtained culture supernatant with a protein A column to obtain antibody solutions containing each anti-IV collagen 7S domain monoclonal antibody and anti-IV collagen monoclonal antibody, respectively. The protein concentration of each antibody solution was determined from the absorbance at a wavelength of 280 nm.

[0135] (3) Confirmation of the reaction specificity of monoclonal antibodies

[0136] The type IV collagen 7S fragment and pepsin-soluble type IV collagen fragment prepared in (1) above were used as antigens, diluted to 1 μg / mL with PBS, and dispensed into a 96-well plate at 100 μL / well. After each antigen was adsorbed overnight at 4°C, the plate was blocked in the blocking buffer for two hours. Next, each antibody solution obtained in (2) was diluted stepwise to 1000 ng / mL to 3.9 ng / mL and dispensed at 100 μL / well and cultured for 1 hour. After washing 4 times with the washing buffer, 100 μL / well of a 5000-fold diluted POD-Goat anti-mouse IgG Fcγ was added to each well. After culturing for 30 minutes, each well was washed 4 times with the washing buffer and colored with TMB solution. The color development reaction was stopped with H2SO4, and the absorbance of each well was measured with an enzyme reader.

[0137] Respectively, Figure 2 The results show the relationship between the concentration of each monoclonal antibody (Ab concentration (ng / mL)) and the absorbance (the ratio of wavelength 450nm to 630nm (A450 / 630), the same below) when the type IV collagen 7S fragment prepared in (1) is used as an antigen. Figure 3 The results of the relationship between the concentration of each monoclonal antibody (Ab concentration (ng / mL)) and the absorbance when the pepsin-solubilized type IV collagen fragment was used as the antigen were shown. It was confirmed that: monoclonal antibodies that specifically bind to the type IV collagen 7S fragment obtained by the collagenase treatment (CIV09, CIV13: anti-type IV collagen 7S domain monoclonal antibodies) and monoclonal antibodies that bind to the pepsin-solubilized type IV collagen fragment but not to the type IV collagen 7S fragment (CIV03: anti-type IV collagen monoclonal antibody) were obtained.

[0138] (4) Preparation of Antibody-Bound Particles

[0139] Using a carboxyl-amine crosslinking agent (carbodiimide, Thermo-Fisher Scientific), according to the product manual, the anti-IV collagen 7S domain monoclonal antibody CIV09 (hereinafter, referred to as "CIV09 antibody" as the case may be) was chemically bound to magnetic particles (manufactured by FUJIREBIO) to obtain CIV09 antibody solid-phase magnetic particles. Similarly, the anti-IV collagen monoclonal antibody CIV03 (hereinafter, referred to as "CIV03 antibody" as the case may be) was chemically bound to magnetic particles to obtain CIV03 antibody solid-phase magnetic particles.

[0140] (5) Preparation of labeled antibodies

[0141] The anti-type IV collagen 7S domain monoclonal antibody CIV13 (hereinafter referred to as "CIV13 antibody" depending on the situation) was combined with 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, first, according to convention, the desalted CIV13 antibody and pepsin are mixed in 0.1M citric acid buffer (pH 3.5), and the mixture is allowed to stand at 37°C for 1 hour for pepsin digestion. After the reaction is stopped, gel filtration purification is performed to obtain the CIV13 antibody with the Fc region removed. Next, 2-mercaptoethylamine hydrochloride is added for thiolation. It is then desalted to obtain the Fab' fragment of the CIV13 antibody.

[0142] Alkaline phosphatase treated with N-(4-maleimidobutyryl) succinimide (GMBS) was mixed with the Fab' fragment of the CIV13 antibody for coupling. 2-Mercaptoethylamine hydrochloride and iodoacetamide were added to the coupling solution to stop the reaction, thereby obtaining an alkaline phosphatase-labeled CIV13 antibody (ALP-labeled CIV13 antibody).

[0143] (6) Study on salt concentration

[0144] First, 30 μL of the sample was added to 50 μL of a particle suspension (50 mM Tris, 1% BSA, 0.15 M to 1.5 M NaCl) containing 0.03% of the CIV09 antibody immobilized magnetic particles prepared in (4) and having different salt concentrations, and the mixture was reacted at 37°C for 8 minutes. As the sample, serum from patients with cirrhosis, non-alcoholic fatty hepatitis (liver disease) or liver cancer (patient sample) and serum from healthy subjects (healthy subject sample) were used, as well as the type IV collagen 7S fragment (Type IV COL 7S) prepared in (1) above, pepsin-solubilized type IV collagen fragment (Type IV COL), and collagenase partially digested type IV collagen fragment (partially digested Type IV COL) diluted to 10 ng / mL with Tris buffer (50 mM Tris, 2% BSA, 0.15 M NaCl, pH 7.2). When the particle suspension contained 0.15M, 0.3M, 0.6M, 1.0M, 1.2M and 1.5M NaCl, the salt concentrations of the reaction systems to which the samples were added were 0.15M, 0.24M, 0.43M, 0.68M, 0.80M and 0.99M, respectively.

[0145] After the reaction, B / F separation was performed using a magnet and the sample was washed with Lumipulse (registered trademark) washing solution (manufactured by FUJIREBIO). Then, 50 μL of a labeled fluid (50 mM Bis-Tris, 600 mM NaCl, 2% BSA, 1 mM MgCl2, 0.3 mM ZnCl2) containing 1 μg / mL of the ALP-labeled CIV13 antibody prepared in the above (5) was added and reacted at 37°C for 8 minutes. After B / F separation with a magnet and washing with Lumipulse washing solution, 200 μL of Lumipulse (registered trademark) substrate solution (manufactured by FUJIREBIO) containing 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)benzene-1,2-dioxetane disodium salt (AMPPD) was added, and the enzyme reaction was carried out at 37°C for 4 minutes, and the luminescence at a wavelength of 463 nm was measured. Similarly, a standard solution (50 mM Tris, 0.15 M NaCl, 2% BSA, pH 7.2) containing a known concentration of the type IV collagen 7S fragment was measured to prepare a standard curve. Using the prepared standard curve, the concentration of the human type IV collagen fragment containing the 7S domain in each sample was calculated from the luminescence of each sample.

[0146] The results are shown in Tables 1 to 3 below. In Tables 1 to 3, the salt concentration represents the salt concentration of the particle suspension, and each value represents the ratio ("%") of the luminescence amount under each condition relative to the luminescence amount when the salt concentration of the particle suspension is 0.15M. In addition, in Table 1, each value of the patient sample represents the average value of 15 samples from liver disease patients (liver disease average), and each value of the healthy person sample represents the average value of 130 samples from healthy people (healthy person average). On the other hand, in Table 3, the patient sample and the healthy person sample each represent the values ​​of 5 of each sample (samples 1 to 5).

[0147] [Table 1]

[0148] Salt concentration 0.15M 0.6M 1.0M 1.2M 1.5M Type IV COL 7S 100％ 104％ 101％ 103％ 106％ Liver disease average 100％ 90％ 90％ 91％ 90％ Average healthy people 100％ 83％ 80％ 80％ 76％

[0149] [Table 2]

[0150] Salt concentration 0.6M 1.0M 1.2M 1.5M Type IV COL 105％ 123％ 132％ 164％ Partial digestion of Type IV COL 104％ 114％ 118％ 117％

[0151] [Table 3]

[0152]

[0153] As shown in Table 1 and Table 3, when the sample is type IV collagen 7S fragment (Type IV COL 7S), no change in luminescence due to changes in salt concentration was found. On the other hand, as shown in Table 3, if the salt concentration is set to 0.3M (0.24M in terms of the salt concentration of the reaction system), the luminescence of both the patient sample and the healthy sample decreases, and the healthy sample decreases particularly. Further, if the salt concentration is increased to 0.6M to 1.0M (0.43M to 0.68M in terms of the salt concentration of the reaction system), the luminescence of the patient sample increases, but in the healthy sample, as the salt concentration increases, the luminescence decreases, and this tendency is significantly increased relative to the patient sample. Moreover, as shown in Table 1, when the salt concentration is 0.6M to 1.5M (0.43M to 0.99M in terms of the salt concentration of the reaction system), the luminescence in the healthy sample decreases significantly. This shows that at high salt concentrations, nonspecific reactions in healthy samples are particularly reduced.

[0154] In addition, as shown in Tables 1 and 2, with respect to the type IV collagen 7S fragment, in which no change in luminescence intensity was observed due to a change in salt concentration, when the pepsin-solubilized type IV collagen fragment (Type IV COL) and the collagenase-partially digested type IV collagen fragment (Partially digested Type IV COL) were used as samples, the luminescence intensity increased as the salt concentration increased when the salt concentration was 0.6 M (0.43 M in terms of the salt concentration of the reaction system). This is considered to be because the 7S domain is shielded in the fragment containing part or all of the TH region except the type IV collagen 7S fragment contained in the pepsin-solubilized type IV collagen fragment or the collagenase-partially digested type IV collagen fragment, and the shielding is released by the increase in salt concentration.

[0155] It is therefore believed that in the sample, the human type IV collagen fragment containing the 7S domain varies in various ways, such as the pepsin-solubilized type IV collagen fragment or the fragment containing the TH region domain contained in the collagen fragment partially digested with collagenase. In contrast, by increasing the salt concentration, the exposure reaches a certain level, allowing a more accurate measurement value to be obtained.

[0156] From the above, it was confirmed that, in particular, by making the salt concentration of the reaction system above 0.35 M (for example, above 0.43 M (salt concentration of the particle suspension 0.6 M)), the nonspecific reaction in healthy human samples was particularly reduced in the sandwich immunoassay of the present invention using a combination of anti-type IV collagen 7S domain monoclonal antibody solid-phase magnetic particles and labeled anti-type IV collagen 7S domain monoclonal antibodies.

[0157] (7) Study on the sensitivity of the measurement system

[0158] As a sample, the type 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 dilution series of standard solutions. The luminescence amount 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 sample was used. In addition, the concentration of the human type IV collagen fragment containing the 7S domain in each sample was calculated using a standard curve. In the measurement, each low concentration standard solution (less than 2.0 ng / mL) was measured 20 times, and for the others, the measurement was repeated twice.

[0159] Figure 4 The results of the relationship between the concentration of type IV collagen 7S fragment (7S concentration (ng / mL)) and the luminescence amount (signal, average value) in the standard solution are shown in FIG. Figure 5 The results of the relationship between the actual concentration of the type IV collagen 7S fragment in the standard solution (7S concentration (ng / mL)) and the coefficient of variation (CV value (%), average value) of the concentration of the human type IV collagen fragment containing the 7S domain quantified by the standard curve (quantitative value) are shown in the figure. The detection limit is set to: the concentration at which the average value of the measured counts when the value is 0 ng / mL lower than the average value - 2SD (SD: standard deviation) of the luminescence amount (signal) of the low-concentration standard solution + 2SD. In addition, the quantitative limit is a concentration at which the coefficient of variation (CV value (%)) is less than 10%. According to Figure 4 , the detection limit is below 0.1ng / mL, according to Figure 5 The limit of quantification was below 0.3 ng / mL.

[0160] <Comparison with the existing method (comparative example)>

[0161] (Example 2)

[0162] As samples, 30 sera from healthy subjects (healthy subject group), 56 sera from cirrhotic patients (cirrhotic subject group), and 38 sera from NASH patients (NASH subject group) were used. Except for using these samples, the luminescence intensity was measured in the same manner as in (7) of Example 1, and the concentration of the fragment containing the type IV collagen 7S domain in each sample was calculated using a standard curve.

[0163] (Comparative Example 1)

[0164] For the same samples as in Example 2, a Type IV collagen·7S kit (manufactured by DENIS Pharma, hereinafter referred to as the "RIA kit" as the case may be) which is a radioimmunoassay using an anti-human type IV collagen rabbit polyclonal antibody was used, and the concentration of the fragment containing the type IV collagen 7S domain in each sample was calculated according to the attached file. According to the RIA kit, the fragment containing the type IV collagen 7S domain in the sample (in the RIA kit, "type IV collagen·7S", 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, if a fragment containing the iodinated type IV collagen 7S domain (in the RIA kit, "type IV collagen·7S( 125 I)”) (labeled antigen), the anti-human type IV collagen rabbit polyclonal antibody that has not bound to the target substance in the sample reacts with the labeled antigen to form a labeled antigen-anti-human type IV collagen rabbit polyclonal antibody complex (complex 2). Anti-rabbit γ-globulin goat serum (goat antibody) is added thereto, and the goat antibody reacts with complex 2 to form a labeled antigen-anti-human type IV collagen rabbit polyclonal antibody-goat antibody complex, which is precipitated. Thus, by measuring the radioactivity of the precipitate after removing the unreacted labeled antigen, the concentration of the target substance (a fragment containing the type IV collagen 7S domain) can be determined.

[0165] (Relevance)

[0166] exist Figure 6 The correlation between the measurement results of Example 2 (CL 7S: the concentration of the fragment containing the type IV collagen 7S domain in the sample (ng / mL)) and the measurement results of Comparative Example 1 (RIA 7S: the concentration of the fragment containing the type IV collagen 7S domain in the sample (ng / mL)) is shown. Figure 7 Only the correlations for the healthy group are shown in Figure 6 The following LC51 and LC28 (i.e., cirrhosis group: 54 cases) are not included. Figure 6 As shown in FIG. 1 , the results of the sandwich immunoassay based on the combination of the anti-IV collagen 7S domain monoclonal antibody immobilized magnetic particles and the labeled anti-IV collagen 7S domain monoclonal antibody of the present invention (CL 7S) and the results of the RIA kit (RIA 7S) were highly correlated, with a correlation coefficient of 0.947. Figure 7As shown, the correlation coefficient is as low as 0.581 only in the healthy group, and it can be seen that according to the sandwich immunoassay of the present invention, the measured value of the healthy sample is significantly reduced. That is, according to the sandwich immunoassay using the monoclonal antibody of the present invention, the measured values ​​of the cirrhosis group and the NASH group are not reduced, but only the measured value of the healthy group can be reduced, and compared with the conventional RIA kit, the patient group and the healthy group can be distinguished with higher accuracy.

[0167] (Evaluation of diagnostic ability 1)

[0168] (Example 3)

[0169] As samples, 25 samples of serum from healthy subjects (healthy subject group 1) and 30 samples of serum from 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, and the concentration of the fragment containing the type IV collagen 7S domain in each sample was calculated using a standard curve.

[0170] (Comparative Example 2)

[0171] The concentration of the fragment containing the type IV collagen 7S domain in each sample was calculated using the RIA kit in the same manner as in Comparative Example 1 except that the same samples as in Example 3 were used.

[0172] (distributed)

[0173] exist Figure 8 The distribution of the measurement results (measured value: concentration of the fragment containing the type IV collagen 7S domain in the sample (ng / mL)) of Example 3 ((b) CL 7S) and Comparative Example 2 ((a) RIA 7S) for the healthy group 1 is shown in FIG. Fig. 9 The distribution of the measurement results of Example 3 ((b) CL 7S) and Comparative Example 2 ((a) RIA 7S) for the healthy subject group 2 is shown in FIG.

[0174] like Figures 8-9 As shown, it was confirmed that in the sandwich immunoassay using the monoclonal antibody of the present invention, the measured values ​​were significantly lower in both healthy groups compared with the RIA kit, and the false positive rate in the healthy group could be reduced.

[0175] (Evaluation of diagnostic ability 2)

[0176] The diagnostic accuracy of Example 2 (CL 7S) and Comparative Example 1 (RIA 7S) was compared by ROC (Receiver Operating Characteristic) analysis. Fig.10The ROC curves (FPR (false positive rate, 1-specificity) and TPR (positive rate, sensitivity) curves) of the cirrhosis group are shown in Fig.11 The ROC curve of the NASH group is shown in FIG. Fig.10 As shown in FIG. 1 , 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 sandwich immunoassay using the monoclonal antibody of the present invention has a particularly high diagnostic accuracy for cirrhosis. Fig.11 As shown, 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 in the sandwich immunoassay of the present invention is particularly high.

[0177] (The form of type IV collagen that can be measured)

[0178] In the correlation between Example 2 and Comparative Example 1, samples (LC51 and LC28) were found in the liver cirrhosis group where the measured values ​​of Comparative Example 1 were significantly lower than the measured values ​​of Example 2. Therefore, the forms of type IV collagen included in the liver cirrhosis group sample (LC51) with a large deviation, the liver cirrhosis group sample (LC58) with a relatively small deviation in measured values, and the healthy person group sample (healthy person sample) were compared.

[0179] (Example 4)

[0180] First, 500 μL of each sample was loaded onto a Superose 6FPLC 10 / 300 equilibrated with a fractionation buffer (50 mM Tris, 150 mM NaCl, 0.05% CHAPS, 0.09% NaN3, pH 7.2), and gel filtration was performed using the fractionation buffer at 0.5 mL / min. The fractionation was performed in 0.25 mL fractions, and the luminescence (CL signal) of each fraction was measured as a sample in the same manner as in (7) of Example 1. In addition, as a control sample, the type IV collagen 7S fragment prepared in (1) was also fractionated and measured in the same manner.

[0181] (Comparative Example 3)

[0182] As a comparative object, the luminescence amount 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.

[0183] [result]

[0184] Respectively, in Fig.12 (a) shows the luminescence intensity (solid line) of each fraction of sample LC58 when CIV09 antibody-immobilized magnetic particles (Example 4) are used. Fig.12 (b) shows the luminescence intensity (solid line) of each fraction of sample LC51 when CIV09 antibody-immobilized magnetic particles (Example 4) are used. Fig.12 (c) shows the luminescence intensity (solid line) of each fraction of a healthy human sample when CIV09 antibody-immobilized magnetic particles (Example 4) were used. Fig.12 In (a) to (b), the luminescence intensity of each fraction of type IV collagen 7S fragment (Type IV COL 7S, dotted line) is also shown. Fig.13 (a) shows the luminescence intensity of each fraction of sample LC58 when CIV09 antibody solid-phase magnetic particles are used (Example 4, solid line) or when CIV03 antibody solid-phase magnetic particles are used (Comparative Example 3, dotted line). Fig.13 (b) shows the luminescence amount of each fraction of the type IV collagen 7S fragment.

[0185] like Fig.12 As shown in (a), in sample LC58, signals were detected only in the high molecular weight fraction. Fig.12 As shown in (b), in sample LC51, which has a large deviation between Example 2 and Comparative Example 1, in addition to the high molecular weight fraction, a signal was also detected in a fraction having a molecular weight equivalent to the type IV collagen 7S fragment obtained by collagenase treatment. Fig.13 As shown in (a) to (b), when the measurement was performed using CIV03 antibody-immobilized magnetic particles, no signal could be detected from any fraction in the type IV collagen 7S fragment ( Fig.13 (b)). Similarly, no signal could be detected in the fraction corresponding to the type IV collagen 7S fragment in sample LC58 ( Fig.13 (a)).

[0186] That is, it was shown that, for comprehensively measuring "fragments containing the type IV collagen 7S domain" in samples from patients with liver cirrhosis and the like, including small-molecule type IV collagen 7S fragments, a sandwich immunoassay using a monoclonal antibody that binds to pepsin-solubilized type IV collagen fragments but does not bind to type IV collagen 7S fragments (e.g., CIV03 antibody: Comparative Example 3) as at least a capture antibody or a labeling antibody is insufficient. Therefore, it was shown that, by using a monoclonal antibody specific to the type IV collagen 7S domain for either the capture antibody or the labeling antibody in the sandwich immunoassay of the present invention, it is possible to measure fragments containing the type IV collagen 7S domain in samples from patients with liver cirrhosis and the like with higher accuracy.

[0187] (Quantitative evaluation)

[0188] Dilution linearity was examined for samples whose measured values ​​deviated between Example 2 and Comparative Example 1. When a measured value was obtained by a specific reaction, if the sample was diluted and measured, a lower measured value could be obtained depending on the dilution rate, and thus dilution linearity could be confirmed.

[0189] (Example 5)

[0190] As samples, each sample of the cirrhosis group was diluted 5-fold or 10-fold with a standard solution diluent (50 mM Tris, 0.15 M NaCl, 2% BSA, pH 7.2) to obtain a dilution. Except for using these samples, the luminescence amount was measured in the same manner as in Example 2, and the concentration of the fragment containing the type IV collagen 7S domain in each sample was calculated using a standard curve.

[0191] (Comparative Example 4)

[0192] The concentration of the fragment containing the type IV collagen 7S domain in each sample was calculated using the RIA kit in the same manner as in Comparative Example 1, except that the same samples as in Example 5 were used.

[0193] [result]

[0194] For LC27, LC28, LC37, LC51 and LC58 in the samples of the cirrhosis group, including LC51 and LC28 that deviated greatly between Example 2 and Comparative Example 1, the measured values ​​and their recovery rates (measured value × dilution factor / measured value at dilution factor 1 × 100 (%)) in Example 5 (CL 7S) and Comparative Example 4 (RIA 7S) are shown in the following Table 4, respectively.

[0195] [Table 4]

[0196]

[0197] As shown in Table 4, in any sample, the recovery rate of the sandwich immunoassay using the monoclonal antibody of the present invention was almost 100%, and dilution linearity (quantitativeness) was confirmed, whereas the RIA kit could not obtain dilution linearity.

[0198] (Analysis of samples with deviation in measured values)

[0199] (Example 6)

[0200] For the sample LC51 whose measured value deviates the most between the embodiment 2 and the comparative example 1, gel filtration is performed in the same manner as described above (the form of type IV collagen that can be measured), and the luminescence amount (signal) of each fraction is measured. In addition, except for using CIV03 antibody-immobilized magnetic particles as antibody-immobilized magnetic particles for control, the luminescence amount of each fraction is measured in the same manner. Furthermore, the obtained fractions are divided into pools 1 to 6, and the luminescence amount (signal) of each pool is measured in the same manner as (7) of embodiment 1. On the other hand, the sample LC51 before gel filtration is also measured in the same manner as a direct measurement value.

[0201] (Comparative Example 5)

[0202] The luminescence amount of each cell was measured using the RIA kit in the same manner as in Comparative Example 1, except that the same cell as in Example 6 was used. On the other hand, the sample LC51 before gel filtration was measured in the same manner as well, and the result was taken as a direct measurement value.

[0203] [result]

[0204] exist Fig.14 The luminescence intensity of each fraction when CIV09 antibody was immobilized on magnetic particles (Example 6, solid line) and the luminescence intensity of each fraction when CIV03 antibody was immobilized on magnetic particles (dashed line) for sample LC51 are shown in the figure. In addition, the concentration of the fragment containing the type IV collagen 7S domain in each pool was calculated from the measured value of each pool as the pool recovery value (ng / mL). Fig.15 The recovery values ​​of each pool in the sandwich immunoassay using a monoclonal antibody (Example 6, CL7S, solid line) and the recovery values ​​of each pool when using an RIA kit (Comparative Example 5, RIA 7S, dotted line) for sample LC51 are shown.

[0205] In addition, the total of the pool recovery values ​​of pools 1 to 6 was taken as the recovery value, and the following formula was used based on each direct measurement value: Recovery rate (%) = Recovery value / Direct measurement value × 100. The result of calculating the recovery rate was 87% for the sandwich immunoassay (Example 6, CL 7S) using the monoclonal antibody of the present invention. In contrast, the direct measurement value of the RIA kit (Comparative Example 5, RIA) was low, with a recovery rate of 237%, far exceeding 100%. Therefore, it is speculated that the CIV03 antibody or the anti-human type IV collagen polyclonal antibody used in the RIA kit is greatly affected by substances present in the sample or sites other than the 7S domain of type IV collagen, and it is believed that the measurement using these cannot give correct values. On the other hand, it is shown that in the sandwich immunoassay using the monoclonal antibody of the present invention, a fragment containing the 7S domain of type IV collagen can be specifically detected.

[0206] Industrial Applicability

[0207] According to the present invention, a measurement method and a kit therefor can be provided, which will not reduce the specificity for samples derived from patients (positive samples), can reduce the nonspecificity for samples derived from healthy persons (negative samples), and can measure fragments containing the human type IV collagen 7S domain in the sample with high accuracy.

[0208] Fragments containing the 7S domain of human type IV collagen in the blood are mainly used as biomarkers for diagnosing cirrhosis or non-alcoholic steatohepatitis (NASH). By using the present invention for auxiliary diagnosis, the diagnostic accuracy can be improved and the non-specificity for samples from healthy persons can be substantially reduced, thereby enabling screening and identification of patients in the early stages of liver fibrosis.

Claims

1. A method for capturing a fragment containing the 7S domain of human type IV collagen in a sample using a capture antibody, characterized in that: The capture antibody is formed by fixing a first monoclonal antibody capable of specifically binding to the 7S domain of human type IV collagen on a carrier. In the method, a sample containing a fragment comprising the 7S domain of human type IV collagen is contacted with the capture antibody. The salt concentration of the reaction system of the fragment comprising the human type IV collagen 7S domain and the first monoclonal antibody is 0.42 mol / L to 1.0 mol / L.

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