WO2018016607A1 - METHOD FOR TESTING FOR IgG4-RELATED DISEASE - Google Patents

METHOD FOR TESTING FOR IgG4-RELATED DISEASE Download PDF

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WO2018016607A1
WO2018016607A1 PCT/JP2017/026359 JP2017026359W WO2018016607A1 WO 2018016607 A1 WO2018016607 A1 WO 2018016607A1 JP 2017026359 W JP2017026359 W JP 2017026359W WO 2018016607 A1 WO2018016607 A1 WO 2018016607A1
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antibody
laminin
igg4
integrin
related disease
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PCT/JP2017/026359
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French (fr)
Japanese (ja)
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雅広 塩川
裕三 児玉
千葉　勉
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国立大学法人京都大学
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Priority to JP2018528879A priority Critical patent/JP6970975B2/en
Publication of WO2018016607A1 publication Critical patent/WO2018016607A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/067—Pancreatitis or colitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a test method for IgG4-related diseases, a test reagent for testing IgG4-related diseases, a method for evaluating the effect of treatment on IgG4-related diseases, and a method for screening candidate substances for therapeutic agents for IgG4-related diseases. .
  • IgG4-related diseases are disease groups characterized by tissue infiltration or tumor formation of high serum IgG4 levels and IgG4-positive plasma cells.
  • IgG4-related disease is a relatively new disease concept, and the cause is unknown so far.
  • affected organs pancreas, bile duct, lacrimal gland, salivary gland, central nervous system, thyroid gland, lung, liver, gastrointestinal tract, kidney, prostate, retroperitoneum, artery, lymph node, skin, mammary gland and the like are known.
  • Lesions often have features in multiple organs and as systemic disease, but may be single organ lesions.
  • AIP Autoimmune pancreatitis
  • IgG4-related kidney disease IgG4-related lacrimal salivary glanditis and the like are known as diseases classified as IgG4-related diseases.
  • IgG4-related diseases The cause of IgG4-related diseases has not been known so far, and a marker substance that serves as an indicator of IgG4-related diseases has not been known. For this reason, it was not easy to diagnose it as an IgG4-related disease.
  • AIP is characterized by tissue infiltration in the pancreas, it is difficult to distinguish it from pancreatic cancer from the observed image of the tissue, and AIP may be erroneously diagnosed as pancreatic cancer.
  • methods for detecting autoimmune pancreatitis include a method for detecting an antibody that immunologically reacts with HSP10 (Patent Document 1) and a method for detecting an antibody that immunologically reacts with amylase ⁇ 2-A (patent 1).
  • Patent Document 1 a method for detecting an antibody that immunologically reacts with HSP10
  • Patent Document 2 a method for detecting an antibody that immunologically reacts with amylase ⁇ 2-A
  • pathogen of IgG4-related disease can be identified, testing for IgG4-related disease using the pathogen as an index, creating a model animal of IgG4-related disease using the pathogen, and developing a new therapeutic drug targeting the pathogen And so on.
  • pathogenic substances for IgG4-related diseases have not been elucidated.
  • the present inventors have surprisingly clarified that the pathogen of IgG4-related disease is an autoantibody against laminin, and that anti-laminin antibody and anti-integrin antibody in serum are indicators of IgG4-related disease. Based on this finding, the inventors have completed the following invention.
  • a test method for IgG4-related diseases Anti-laminin antibody detection step for detecting an antibody that immunologically reacts with laminin as an indicator of IgG4-related disease, and / or immunological reaction with integrin in a sample as an indicator of IgG4-related disease
  • a method comprising an anti-integrin antibody detection step of detecting an antibody to be detected.
  • the IgG4-related disease is autoimmune pancreatitis
  • the antibody in the anti-laminin antibody detection step is an antibody that immunologically reacts with laminin-511.
  • the IgG4-related disease is autoimmune pancreatitis
  • the antibody in the anti-integrin antibody detection step is an antibody that immunologically reacts with integrin ⁇ 6 ⁇ 1.
  • the IgG4-related disease is IgG4-related kidney disease, The method according to (1), wherein the antibody in the anti-laminin antibody detection step is an antibody that immunologically reacts with laminin-521.
  • the anti-laminin antibody detection step comprises detecting the antibody using laminin as an antigen.
  • the anti-integrin antibody detection step includes detecting the antibody using integrin as an antigen.
  • the specimen is a blood sample.
  • a test method for autoimmune pancreatitis As an index of autoimmune pancreatitis, an anti-laminin antibody detection step for detecting an antibody immunologically reactive with laminin-511 in a sample blood sample, and as an index of autoimmune pancreatitis in a sample blood sample And an anti-integrin antibody detection step of detecting an antibody that immunologically reacts with integrin ⁇ 6 ⁇ 1.
  • a test reagent for testing IgG4-related diseases, including laminin. The IgG4-related disease is autoimmune pancreatitis, The test reagent according to (8), wherein the laminin is laminin-511.
  • the IgG4-related disease is IgG4-related kidney disease, The test reagent according to (8), wherein the laminin is laminin-521.
  • the IgG4-related disease is autoimmune pancreatitis, The test reagent according to (12), wherein the integrin is integrin ⁇ 6 ⁇ 1.
  • a test reagent for autoimmune pancreatitis comprising laminin-511 and integrin ⁇ 6 ⁇ 1 in a form immobilized on a solid phase.
  • a method for evaluating the effect of treatment on an IgG4-related disease An anti-laminin antibody detection step of detecting an antibody that immunologically reacts with laminin in a specimen obtained from a test animal treated for an IgG4-related disease; and / or A method comprising an anti-integrin antibody detection step of detecting an antibody that immunologically reacts with an integrin in a specimen obtained from a test animal treated for an IgG4-related disease.
  • a method for screening candidate substances for therapeutic agents for IgG4-related diseases An anti-laminin antibody detection step for detecting an antibody that immunologically reacts with laminin in a specimen obtained from an animal on which a test substance has been allowed to act; And a selection step of selecting the test substance as a candidate substance for a therapeutic drug for IgG4-related disease when the antibody in the specimen decreases due to the action of the test substance.
  • a method for screening candidate substances for therapeutic agents for IgG4-related diseases An anti-integrin antibody detection step for detecting an antibody that immunologically reacts with an integrin in a sample obtained from an animal on which a test substance has been allowed to act; And a selection step of selecting the test substance as a candidate substance for a therapeutic drug for IgG4-related disease when the antibody in the specimen decreases due to the action of the test substance.
  • a method for producing a non-human model animal of an IgG4-related disease A method comprising at least one of an anti-laminin antibody administration step of administering an antibody that immunologically reacts with laminin to a non-human animal, and a laminin immunization step of immunizing a non-human animal with laminin as an antigen.
  • a method for producing a non-human animal model of an IgG4-related disease A method comprising at least one of an anti-integrin antibody administration step of administering an antibody that immunologically reacts with an integrin to a non-human animal, and an integrin immunization step of immunizing the non-human animal with an integrin as an antigen.
  • a method for obtaining an index of an IgG4-related disease A method comprising an anti-laminin antibody detection step of detecting an antibody immunologically reactive with laminin in a sample, and / or an anti-integrin antibody detection step of detecting an antibody immunologically reactive with integrin in the sample.
  • the IgG4-related disease is autoimmune pancreatitis
  • the method according to (20), wherein the antibody in the anti-laminin antibody detection step is an antibody that immunologically reacts with laminin-511.
  • the IgG4-related disease is autoimmune pancreatitis, The method according to (20) or (21), wherein the antibody in the anti-integrin antibody detection step is an antibody that immunologically reacts with integrin ⁇ 6 ⁇ 1.
  • the IgG4-related disease is IgG4-related kidney disease;
  • the method according to (20), wherein the antibody in the anti-laminin antibody detection step is an antibody that immunologically reacts with laminin-521.
  • the method according to any one of (20) to (23), wherein the anti-laminin antibody detection step comprises detecting the antibody using laminin as an antigen.
  • the IgG4-related disease is autoimmune pancreatitis, The use according to (27), wherein the laminin is laminin-511.
  • the IgG4-related disease is IgG4-related kidney disease, The use according to (27), wherein the laminin is laminin-521.
  • (31) Use of an integrin in the manufacture of a test reagent for testing an IgG4-related disease.
  • the IgG4-related disease is autoimmune pancreatitis, The use according to (31), wherein the integrin is integrin ⁇ 6 ⁇ 1.
  • a method for testing an IgG4-related disease a test reagent for testing an IgG4-related disease, a method for evaluating the effect of a treatment on an IgG4-related disease, a therapeutic agent for an IgG4-related disease, An effective means is provided as a method for screening candidate substances.
  • FIG. 1A shows H & E staining of pancreas 12 hours after IgG administration (upper row) and Gr1 staining (lower row) of control IgG-treated mice (left column) and patient IgG-treated mice (right column) in Experiment 1. ) Result.
  • the scale bar in the observed image shows 50 ⁇ m in the upper stage and 20 ⁇ m in the lower stage.
  • FIG. 1B shows the correlation between the dose of control IgG (triangle) and patient IgG (circle) and the edema area in Experiment 1.
  • FIG. 1A shows H & E staining of pancreas 12 hours after IgG administration (upper row) and Gr1 staining (lower row) of control IgG-treated mice (left column) and patient IgG-treated mice (right column) in Experiment 1.
  • the scale bar in the observed image shows 50 ⁇ m in the upper stage and 20 ⁇ m in the lower stage.
  • FIG. 1B shows the correlation between the dose of
  • FIG. 1C shows the histopathological grade of necrosis or hemorrhage of pancreas 12 hours after IgG administration and the number of Gr1 positive cells in control IgG-treated mice (left) and patient IgG-treated mice (right) in Experiment 1.
  • FIG. 2A shows IgG in the pancreas 12 hours after IgG administration (upper row), IgG1 (middle row), IgG4 of control IgG-treated mice (left column) and patient IgG-treated mice (right column) in Experiment 1.
  • the result of immunohistochemical staining of (lower) is shown.
  • the scale bar in the observed image indicates 20 ⁇ m.
  • FIG. 2B shows the results of immunofluorescence staining of pancreatic tissue in control IgG-treated mice and patient IgG-treated mice in Experiment 1.
  • the scale bar in the observed image indicates 20 ⁇ m.
  • Control 1 in the upper part of FIG. 3A shows pancreatic H & E 12 hours after administration of mice administered with one or both of control IgG1 and control IgG4 obtained from a specific one of the 10 controls (control 1). It is an observation image of dyeing
  • the scale bar in the observed image indicates 20 ⁇ m.
  • FIG. 3A shows 12 hours after administration of a mouse administered with one or both of patient IgG1 and patient IgG4 from one specific patient (patient 9) among 10 IgG4-related disease patients.
  • 2 is an observation image of H & E staining (middle stage) and an observation image of immunohistochemical staining of Gr1 (lower stage). The scale bar in the observed image indicates 20 ⁇ m.
  • FIG. 3B shows pancreatic necrosis or 12 hours after administration of mice that received one or both of control IgG1 and control IgG4 (left) and mice that received one or both of patient IgG1 and patient IgG4 (right).
  • FIG. 3C shows pancreatic human IgG1 or human 12 hours after administration of mice administered with one or both of patient IgG1 and patient IgG4 from one particular patient (patient 1) out of 10 IgG4-related diseases It is an observation image of immunohistochemical staining of IgG4.
  • the scale bar in the observed image indicates 20 ⁇ m.
  • the upper left of FIG. 4A shows an observation image obtained by H & E staining of a pancreatic tissue section of an AIP patient collected from a boundary portion between a site with AIP lesion (left side) and a site without lesion (right side).
  • FIG. 4A shows an observation image of immunohistochemical staining for IgG4 of the same pancreatic tissue section.
  • the scale bar indicates 200 ⁇ m.
  • the lower left of FIG. 4A is an enlarged image of the lesioned acinar portion in the upper right image of FIG. 4A.
  • the lower part of FIG. 4A is an observation image obtained by enlarging the lesioned leaflet gap portion in the upper right part of FIG. 4A.
  • the lower right side of FIG. 4A is an observation image obtained by enlarging a normal acinar portion without a lesion in the upper right image of FIG. 4A.
  • the scale bar indicates 20 ⁇ m.
  • FIG. 4B shows an observation image obtained by immunofluorescent staining of a lesioned acinus in the pancreas of an AIP patient.
  • the scale bar indicates 20 ⁇ m.
  • the upper left of FIG. 4B shows an observed image of IgG4 stained with immunofluorescence in an acinus with lesions in the pancreas of an AIP patient.
  • the upper part of FIG. 4B shows an observation image of amylase stained with immunofluorescence in the same observation area as the upper left part of FIG. 4B.
  • the upper right of FIG. 4B is an image obtained by superimposing the observed image of IgG4 on the upper left of FIG. 4B and the observed image of amylase in the upper part of FIG. 4B.
  • the lower left of FIG. 4B shows an observed image of IgG4 stained with immunofluorescence in an acinus with lesions in the pancreas of an AIP patient.
  • the lower part of FIG. 4B shows an observation image of collagen IV stained with immunofluorescence in the same observation area as the lower left part of FIG. 4B.
  • the lower right of FIG. 4B is an image obtained by superimposing the observed image of IgG4 on the lower left of FIG. 4B and the observed image of collagen IV in the lower part of FIG. 4B.
  • Each image in FIG. 4C shows an observation image obtained by immunofluorescent staining of a lobular space with a lesion in the pancreas of an AIP patient.
  • the scale bar indicates 20 ⁇ m.
  • FIG. 4C shows an observed image of immunofluorescent-stained IgG4 in a lobular space with lesions in the pancreas of an AIP patient.
  • FIG. 4C shows an observation image of collagen IV stained with immunofluorescence in the same observation region as the left of FIG. 4C.
  • the right side of FIG. 4C is an image obtained by superimposing the observed image of IgG4 on the left side of FIG. 4C and the observed image of collagen IV in FIG. 4C.
  • concentration of anti-human laminin-511 active fragment antibody in AIP patient serum and control serum is shown.
  • the concentration of anti-human collagen IV antibody in AIP patient serum and control serum is shown.
  • the concentration of anti-human fibronectin antibody in AIP patient serum and control serum is shown.
  • FIG. 6 shows an observation image of a tissue section of the pancreas stained by H & E staining.
  • FIG. 6 left is an observation image of the pancreas of the control group, and
  • FIG. 6 right is an observation image of the pancreas of the test group immunized with laminin-511.
  • the concentration of anti-human laminin-521 antibody in AIP patient serum and control serum is shown.
  • the left side of FIG. 8 shows an observation image of the kidney of a mouse subcutaneously administered with IgG4 related kidney disease patient IgG
  • the right side of FIG. 8 shows an observation image of human IgG4 staining of the kidney of a mouse subcutaneously administered with control IgG.
  • the upper row shows the human IgG4 staining observation image of the whole kidney, and the lower row shows the human IgG4 staining observation image of the filamentous body.
  • the measurement results of anti-human laminin-511-E8 IgG antibody levels of serum samples from 10 AIP patients and 10 controls in the training group in Experiment 8 are shown. Results of measurement of anti-human laminin-511-E8 IgG antibody levels in serum samples from 41 AIP patients and 102 controls (20 healthy subjects, 82 non-AIP disease patients) in the validation group in Experiment 8 Indicates.
  • the upper part of FIG. 12 is a pancreatic image obtained by contrast CT of an anti-laminin 511-E8 antibody positive AIP patient.
  • FIG. 12 is a pancreatic image obtained by contrast CT of an anti-integrin ⁇ 6 ⁇ 1 antibody-positive AIP patient.
  • the arrow in each image indicates the lesion site.
  • the measurement results of anti-laminin 511-E8 antibody levels in serum before and after treatment with steroid (prednisone) of 5 patients with anti-laminin 511-E8 antibody positive AIP are shown.
  • the IgG4-related disease is a disease group characterized by tissue infiltration or tumor formation of high serum IgG4 and IgG4-positive plasma cells, specifically, autoimmune pancreatitis (AIP), IgG4-related kidney disease, Examples include IgG4-related lacrimal gland salivary gland, IgG4-related sclerosing cholangitis, IgG4-related eye disease, IgG4-related periarteritis, IgG4-related hypophysitis, IgG4-related lung disease, IgG4-related retroperitoneal fibrosis. ⁇ 2.
  • AIP autoimmune pancreatitis
  • IgG4-related kidney disease examples include IgG4-related lacrimal gland salivary gland, IgG4-related sclerosing cholangitis, IgG4-related eye disease, IgG4-related periarteritis, IgG4-related hypophysitis, IgG4-related lung disease, Ig
  • the present invention firstly A test method for IgG4-related diseases, Anti-laminin antibody detection step for detecting an antibody that immunologically reacts with laminin as an indicator of IgG4-related disease, and / or immunological reaction with integrin in a sample as an indicator of IgG4-related disease
  • the present invention relates to a method comprising an anti-integrin antibody detection step of detecting an antibody to be detected.
  • the antibody against laminin is a pathogen of an IgG4-related disease, and the antibody in a specimen obtained from a test animal suffers from an IgG4-related disease. It was completed based on the surprising finding that it is an indicator of what is being done.
  • the binding between laminin and integrin is a target of a pathogen of an IgG4-related disease, and an anti-laminin antibody in a specimen obtained from a test animal and / or It was completed based on the surprising finding that anti-integrin antibodies are indicative of having an IgG4-related disease.
  • test animal targeted by the test method of the present invention is not particularly limited and may be a human or other non-human mammal, but is preferably a human.
  • Specimens used in the test method of the present invention include body fluids collected from test animals. Specifically, in addition to blood samples such as serum, plasma and whole blood, body fluid samples other than blood such as saliva, spinal fluid and urine may be used. Further, not only body fluids but also tissues collected from test animals, for example, tissues collected from disease sites of IgG4-related diseases to be examined (for example, pancreas, kidney, salivary gland, etc.) can be used as specimens. . The specimen is used in the examination method of the present invention in a form separated from the subject animal.
  • Laminin is a huge heterotrimeric molecule consisting of three subunit chains of ⁇ chain, ⁇ chain and ⁇ chain in the natural type. ⁇ 1 to ⁇ 5 are known as ⁇ chains, ⁇ 1 to ⁇ 3 are known as ⁇ chains, and ⁇ 1 to ⁇ 3 are known as ⁇ chains, and combinations thereof include 12 or more isoforms (see Table 1). In the present invention, laminin isoforms are not limited.
  • the laminin to which the antibody to be detected immunologically reacts is selected from one ⁇ chain selected from ⁇ 1 to ⁇ 5, one ⁇ chain selected from ⁇ 1 to ⁇ 3, and ⁇ 1 to ⁇ 3 1
  • It can consist of ⁇ chains of species, specifically, can be any of the 12 types listed in Table 1 or other isoforms, preferably ⁇ chain is ⁇ 5, ⁇ chain is “Laminin-511” in which ⁇ 1 and ⁇ chain are ⁇ 1, and “Laminin-521” in which ⁇ chain is ⁇ 5, ⁇ chain is ⁇ 2, and ⁇ chain is ⁇ 1.
  • the origin of laminin to which the antibody to be detected immunologically reacts is not particularly limited.
  • an IgG4-related disease is usually an autoimmune disease, it must be the same kind of laminin as the test animal.
  • the nucleotide sequence information of the genes encoding the ⁇ chain, ⁇ chain, and ⁇ chain of laminin from mammalian species such as humans and the amino acid sequence information of each chain can be obtained from a known database (GenBank, etc.).
  • Table 2 shows the accession numbers of the chains constituting laminin for main mammals including humans.
  • the laminin having the amino acid sequence shown in Table 2 may be further subjected to post-translational modification to form laminin.
  • the base sequence information and amino acid sequence information of laminin constituent chains of various mammals can also be obtained from known databases (GenBank, etc.).
  • laminin, laminin-511, and laminin-521 are not particularly limited to natural forms in which the ⁇ chain, the ⁇ chain, and the ⁇ chain are all full lengths, respectively. Fragments, fragments of laminin-511, fragments of laminin-521, natural laminin, natural laminin-511, and forms equivalent to natural laminin-521 are included. Therefore, the antibody to be detected may be one that immunologically reacts with a laminin fragment. Examples of the laminin fragment include those in which at least one of the ⁇ chain, ⁇ chain, and ⁇ chain constituting the natural laminin trimer is shorter than the full length.
  • the laminin fragment preferably forms a trimer, and more preferably has integrin binding activity. It can be confirmed that the laminin fragment forms a trimer by subjecting the laminin fragment to SDS-PAGE and detecting the number of bands. It can be confirmed by ELISA or the like that the laminin fragment has integrin binding activity.
  • Examples of the fragment of laminin that forms a trimer and has integrin binding activity include a fragment obtained by digesting natural laminin with a proteolytic enzyme such as elastase, and particularly preferably an E8 fragment.
  • the E8 fragment of laminin is obtained by removing the C-terminal globular (G) domains 4 and 5 from the ⁇ -chain C-terminal fragment ( ⁇ -chain E8), ⁇ -chain C-terminal fragment ( ⁇ -chain E8) and ⁇ -chain.
  • the C-terminal fragment ( ⁇ -chain E8) is a fragment that forms a trimer (Hiroyuki Ido, Aya Nakamura, Reiko Kobayashi, Shunsuke Ito, Shaoliang Li, Sugiko Futaki, and Kiyotoshi Sekiguchi, The Journal of Biological Chemist 11144-11154, 2007).
  • the E8 fragment of laminin can be obtained by enzymatic digestion of full length laminin with pancreatic elastase.
  • An example of a commercially available E8 fragment of laminin is human laminin-511-E8 (892012, manufactured by Nippi Co., Ltd.).
  • Integrin is a heterodimer molecule consisting of two subunit chains of ⁇ chain and ⁇ chain in the natural type. ⁇ 1 to ⁇ 11, ⁇ V, ⁇ X, ⁇ M, ⁇ L, ⁇ D, ⁇ E, ⁇ IIb as ⁇ chains, ⁇ 1 to ⁇ 8 as ⁇ chains, and a plurality of isoforms having different combinations thereof exist.
  • Integrin ⁇ 6 ⁇ 1, integrin ⁇ 3 ⁇ 1, and integrin ⁇ 6 ⁇ 4 are known as integrin isoforms that can be laminin ligands.
  • the integrin isoform is not limited, but is preferably “integrin ⁇ 6 ⁇ 1” in which the ⁇ chain is ⁇ 6 and the ⁇ chain is ⁇ 1.
  • the origin of the integrin to which the antibody to be detected reacts immunologically is not particularly limited.
  • an IgG4-related disease is usually an autoimmune disease, it is an integrin of the same species as the test animal.
  • the nucleotide sequence information of the genes encoding integrin ⁇ chain and ⁇ chain of mammalian species such as humans and the amino acid sequence information of each chain can be obtained from a known database (GenBank, etc.).
  • Table 3 shows the amino acid sequence and base sequence accession number of each chain constituting human integrin. Three splicing variants are known for each of ⁇ 6 chain and ⁇ 1 chain of human integrin.
  • Human integrin having the amino acid sequence shown in Table 3 that has undergone post-translational modification may also form human integrin.
  • the base sequence information and amino acid sequence information of integrin constituent chains of various mammals other than these can also be obtained from known databases (GenBank etc.).
  • the terms integrin, integrin ⁇ 6 ⁇ 1, and integrin ⁇ 3 ⁇ 1 are not particularly limited to forms in which the ⁇ chain and ⁇ chain are each composed of the full length, respectively, and are each an integrin fragment, integrin ⁇ 6 ⁇ 1 fragment, integrin It includes fragments of ⁇ 3 ⁇ 1, natural integrin, natural integrin ⁇ 6 ⁇ 1, and forms equivalent to natural integrin ⁇ 3 ⁇ 1. Therefore, the antibody to be detected may be one that immunologically reacts with the integrin fragment.
  • integrin fragments include those in which at least one of the ⁇ chain and ⁇ chain constituting the integrin dimer is shorter than the full length.
  • the integrin fragment preferably forms a dimer, and more preferably has laminin binding activity.
  • the fact that the integrin fragment forms a dimer can be confirmed by subjecting the integrin fragment to SDS-PAGE and detecting the number of bands. It can be confirmed by ELISA or the like that the integrin fragment has laminin binding activity.
  • Examples of commercially available integrins include recombinant human integrin ⁇ 6 ⁇ 1 (R & D Systems, Minnesota, USA, product number 7809-A6) and recombinant human integrin ⁇ 3 ⁇ 1 (R & D Systems, Minnesota, USA, product number 2840-A3).
  • integrin fragments include Human ITGA6 & ITGB1 (Beijing China, Sino Biological, product number CT013-H2508H-20), which is a commercially available fragment of human integrin ⁇ 6 ⁇ 1.
  • the anti-laminin antibody detection step is a step of detecting an antibody that immunologically reacts with laminin in a sample.
  • the anti-integrin antibody detection step is a step of detecting an antibody that immunologically reacts with the integrin in the specimen.
  • “detection” refers to whether or not there is an antibody that immunologically reacts with laminin or integrin in the sample, and the content of the antibody in the sample is measured. In other words, it is a concept that encompasses “quantitative”.
  • the anti-laminin antibody detection step and the anti-integrin antibody detection step may be any method that can quantitatively or qualitatively detect the antibody in the specimen, and specific embodiments are not particularly limited.
  • immunological techniques can be used, for example, antibody enzyme method (ELISA method), immunoprecipitation method (IPP method), immunoblot method (IB method), latex agglutination method, immunochromatography method, indirect fluorescent antibody Methods (IF method), radioimmunoassay methods (RIA method) and the like can be mentioned.
  • ELISA method antibody enzyme method
  • IPP method immunoprecipitation method
  • IB method immunoblot method
  • latex agglutination method immunochromatography method
  • IF method indirect fluorescent antibody Methods
  • RIA method radioimmunoassay methods
  • An ELISA method that can process many specimens is particularly preferred.
  • laminin or integrin as described in detail above which is the antigen of the antibody
  • the specimen is contacted with the antigen immobilized on the solid phase, and the antigen and specimen
  • the antibody can be detected by detecting an immune complex with the antibody that may be contained therein.
  • the labeling substance that can be used in the detection, the measuring instrument, and the like there are no particular limitations on the method for suppressing the non-specific reaction associated with the ELISA method, the labeling substance that can be used in the detection, the measuring instrument, and the like.
  • a solid phase for immobilizing an antigen a solid phase having an arbitrary shape such as a plate, a bead, or a tube can be used.
  • the amount of the antibody that immunologically reacts with laminin in the anti-laminin antibody detection step, and the amount of the antibody that immunologically reacts with integrin in the anti-integrin antibody detection step is the amount of the immune complex of the antibody and the antigen. It can be determined as a measured value of the label. It is also possible to prepare a calibration curve with a standard sample containing the antibody at a known concentration, and calculate the amount of the antibody from the measured value of the label using the calibration curve.
  • Determination of whether there is an antibody that immunologically reacts with laminin in the anti-laminin antibody detection process (positive / negative determination), and presence of an antibody that reacts immunologically with integrin in the anti-integrin antibody detection process
  • the determination of whether or not to perform is based on the measured value of the antibody in a specimen obtained from an individual animal subject (eg, a human suspected of suffering from an IgG4-related disease). Can be performed by comparing the measured value of the antibody in a sample obtained from an animal individual (for example, a healthy person) who does not suffer from the disease.
  • the measured value of the antibody in the sample obtained from an individual who does not suffer from an IgG4-related disease may be a value obtained by simultaneous measurement or may be a value obtained by measurement in advance. Good.
  • the measured value of the antibody in the specimen obtained from the test animal individual is significantly larger than the measured value of the antibody in the specimen obtained from the animal individual not suffering from IgG4-related disease, it is judged as positive be able to.
  • the class of the antibody that immunologically reacts with laminin in the anti-laminin antibody detection step and the antibody that immunologically reacts with integrin in the anti-integrin antibody detection step are not particularly limited, but are preferably IgG antibodies.
  • the subclass in IgG antibody is not specifically limited, For example, IgG1 antibody or IgG4 antibody can be detected. Since the present inventors have found that IgG antibodies immunologically reactive with laminin, in particular IgG1 antibodies or IgG4 antibodies, are pathogens in IgG4-related diseases, these are particularly useful as indicators of IgG4-related diseases. Useful.
  • the combination of the type of IgG4-related disease to be examined, the type of antibody that immunologically reacts with laminin, or the type of antibody that immunologically reacts with integrin is not particularly limited.
  • an antibody that immunologically reacts with laminin-511 see Experiment 4
  • an antibody that reacts immunologically with integrin ⁇ 6 ⁇ 1 see Experiment 9 are useful.
  • the antibody that immunologically reacts with laminin-511 is particularly preferably detected by a detection method using a fragment of laminin-511 (particularly, a trimer fragment having integrin binding activity) as an antigen.
  • the antibody that reacts immunologically with integrin ⁇ 6 ⁇ 1 is particularly preferably detected by a detection method using integrin ⁇ 6 ⁇ 1 as an antigen.
  • an antibody that immunologically reacts with laminin-521 is useful as an indicator of IgG4-related kidney disease (see Experiment 6).
  • laminin-511 particularly laminin-511-E8
  • AIP autoimmune pancreatitis
  • the anti-laminin antibody detection step of the IgG4-related disease testing method of the present invention can obtain an index for discriminating between AIP and pancreatic cancer, and is particularly useful.
  • an antibody that immunologically reacts with laminin, particularly laminin-511, particularly laminin-511-E8, in a sample If detected, based on the detection result, it can be determined that the test animal from which the specimen is derived has a low risk of combining a malignant tumor with IgG4-related disease, particularly AIP.
  • an antibody that immunologically reacts with integrin particularly integrin ⁇ 6 ⁇ 1
  • an anti-integrin antibody detection step based on the detection result, It can be determined that the test animal from which the specimen is derived suffers from an IgG4-related disease, particularly AIP.
  • an embodiment including the anti-integrin antibody detection step of the IgG4-related disease test method of the present invention comprises: This is particularly useful because an index for discriminating between AIP and pancreatic cancer can be obtained.
  • an anti-integrin antibody detection step if an antibody immunologically reactive with integrin, particularly integrin ⁇ 6 ⁇ 1, is detected in the sample, the detection result Based on this, it can be determined that the test animal from which the specimen is derived has a high risk of combining malignant tumors with IgG4-related diseases, particularly AIP. It is possible to determine that it is highly necessary to screen a malignant tumor throughout the body for a test animal that has been determined to have a high risk of complication of a malignant tumor.
  • the method for testing an IgG4-related disease of the present invention may include at least one of an anti-laminin antibody detection step and an anti-integrin antibody detection step, and more preferably both an anti-laminin antibody detection step and an anti-integrin antibody detection step.
  • An anti-laminin antibody-negative sample may be anti-integrin antibody-positive
  • an anti-integrin antibody-negative sample may be anti-laminin antibody-positive.
  • Test reagent for IgG4-related disease > The present invention secondly, A test reagent for testing IgG4-related diseases, including laminin, Test reagents for testing IgG4-related diseases, including integrins, or The present invention relates to a test reagent for testing an IgG4-related disease, including laminin and integrin.
  • IgG4-related diseases e.g., laminin, and integrin are as described above for the test method of the present invention.
  • test reagent of the present invention containing laminin can be used in the above-described test method of the present invention including an anti-laminin antibody detection step.
  • test reagent of the present invention containing integrin can be used in the above-described test method of the present invention including an anti-integrin antibody detection step.
  • test reagent of the present invention may contain reagents necessary for immunological measurement, for example, a buffer solution as necessary, and may be provided as a kit comprising a plurality of components.
  • the form of laminin is not particularly limited, and may be a solution state, a dry state, or a form fixed to a solid phase. May be.
  • a buffer solution or a solvent for making it into a solution state before use may be included in the test reagent of the present invention.
  • the form of integrin is not particularly limited, and may be in a solution state, a dry state, or a form fixed on a solid phase. May be.
  • a buffer solution or a solvent for making it into a solution state before use may be included in the test reagent of the present invention.
  • test reagent of the present invention containing laminin in a form immobilized on a solid phase can be in the form of a kit for testing an IgG4-related disease by ELISA.
  • An embodiment of the test reagent of the present invention containing the integrin in a form immobilized on a solid phase can also be in the form of a kit for testing an IgG4-related disease by ELISA.
  • An embodiment of the test reagent of the present invention comprising laminin and integrin in a form immobilized on a solid phase can also be in the form of a kit for testing IgG4-related diseases by ELISA.
  • the form of the solid phase may be any form such as a plate, a bead, or a tube.
  • a kit for examining an IgG4-related disease by ELISA can contain a blocking solution, a washing solution, a sample diluent, an enzyme-labeled secondary antibody, a substrate solution, and the like. ⁇ 4.
  • the present invention A method for assessing the effect of treatment on an IgG4-related disease comprising: An anti-laminin antibody detection step of detecting an antibody that immunologically reacts with laminin in a specimen obtained from a test animal treated for an IgG4-related disease; and / or The present invention relates to a method comprising an anti-integrin antibody detection step of detecting an antibody that immunologically reacts with an integrin in a specimen obtained from a test animal treated for an IgG4-related disease.
  • the above-mentioned “method for evaluating the effect of treatment on an IgG4-related disease” may be expressed as “a method for obtaining an index for evaluating the effect of treatment on an IgG4-related disease”.
  • the antibody that reacts immunologically with laminin and the antibody that reacts immunologically with integrin in the specimen are useful as indicators of IgG4-related diseases. For this reason, the antibody in the specimen obtained from the test animal treated for IgG4-related disease is reduced when the treatment is effective and cured, and when the treatment is not sufficiently effective It remains unchanged or rises from before the start of treatment. Thus, in a sample obtained from a test animal treated for IgG4-related disease. By detecting an antibody immunologically reactive with laminin and / or an antibody immunologically reactive with integrin, it is possible to evaluate the effect of treatment.
  • test animals include humans and non-human animals that have been treated for IgG4-related diseases.
  • IgG4-related diseases includes treatment of IgG4-related diseases such as steroid administration.
  • Laminin in a specimen obtained from a test animal treated with an IgG4-related disease obtained in the anti-laminin antibody detection step and / or anti-integrin antibody detection step in the method for evaluating therapeutic effect of the present invention immunologically Quantitative results of antibodies that react immunologically with reacting antibodies and / or integrins, for example, antibodies that react immunologically with laminin in a specimen obtained from the same animal individual prior to the start of the treatment and / or When the former is smaller than the latter compared with the quantitative result of the antibody that immunologically reacts with integrin, it can be evaluated that the treatment is effective. On the other hand, if the former is about the same as or larger than the latter, it can be evaluated that the treatment is not effective or sufficient. Based on the evaluation result, it is possible to determine whether to continue the treatment, change the treatment policy, or stop the treatment.
  • laminin and immunology in a specimen obtained from a test animal treated with an IgG4-related disease obtained in the anti-laminin antibody detection step and / or anti-integrin antibody detection step in the method for evaluating therapeutic effect of the present invention Quantification results of antibodies that react immunologically and / or antibodies that react immunologically with integrins, for example, antibodies and / or integrins that react immunologically with laminin in specimens obtained from healthy individuals of the same animal species If the former is comparable or smaller than the latter compared to the quantification result of the immunologically reactive antibody, it can be evaluated that the treatment is effective. On the other hand, if the former is larger than the latter, it can be evaluated that the treatment is not effective or sufficient.
  • Method 1 for Screening Candidate Substances for Treatment of IgG4-Related Disease comprising: An anti-laminin antibody detection step for detecting an antibody that immunologically reacts with laminin in a specimen obtained from an animal on which a test substance has been allowed to act; And a selection step of selecting the test substance as a candidate substance for a therapeutic drug for IgG4-related disease when the antibody in the specimen decreases due to the action of the test substance.
  • the antibody that immunologically reacts with laminin in the sample is useful as an indicator of IgG4-related diseases.
  • the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases.
  • the test substance is a test substance that may be a candidate for a new drug, and is not particularly limited.
  • the animal to which the test substance is allowed to act is typically an animal having a large amount of an antibody that immunologically reacts with laminin in a specimen, such as a non-human animal model of an IgG4-related disease. is there.
  • the quantification result of the antibody in the specimen obtained from the animal to which the test substance is applied obtained in the anti-laminin antibody detection step, for example,
  • the former is smaller than the latter compared with the quantification result of the antibody in the specimen obtained from the same animal individual before the test substance is allowed to act,
  • the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases.
  • the quantification result of the antibody in the specimen obtained from the animal to which the test substance is applied, obtained in the anti-laminin antibody detection step is obtained.
  • the former is comparable or smaller than the latter in comparison with the quantification result of the antibody in a sample obtained from a healthy individual of the same animal species, Judging that the antibody in the sample has decreased, the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases. ⁇ 6.
  • the present invention A method for screening candidate substances for therapeutic agents for IgG4-related diseases, comprising: An anti-integrin antibody detection step for detecting an antibody that immunologically reacts with an integrin in a sample obtained from an animal on which a test substance has been allowed to act; And a selection step of selecting the test substance as a candidate substance for a therapeutic drug for IgG4-related disease when the antibody in the specimen decreases due to the action of the test substance.
  • the antibody that immunologically reacts with the integrin in the sample is useful as an indicator of IgG4-related diseases. For this reason, when the antibody in the sample decreases due to the action of a test substance, the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases.
  • the test substance is not particularly limited and is a test substance that may be a candidate for a new drug.
  • the animal to which the test substance is allowed to act is typically an animal having a large amount of an antibody that immunologically reacts with integrin compared to a healthy individual, such as a non-human animal model of an IgG4-related disease. is there.
  • the quantification result of the antibody in the specimen obtained from the animal to which the test substance is applied obtained in the anti-integrin antibody detection step, for example,
  • the former is smaller than the latter compared with the quantification result of the antibody in the specimen obtained from the same animal individual before the test substance is allowed to act,
  • the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases.
  • the quantification result of the antibody in the specimen obtained from the animal to which the test substance is applied, obtained in the anti-integrin antibody detection step is obtained.
  • the former is comparable or smaller than the latter in comparison with the quantification result of the antibody in a sample obtained from a healthy individual of the same animal species, Judging that the antibody in the sample has decreased, the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases. ⁇ 7.
  • Method 1 for producing non-human animal model of IgG4-related disease Sixth, the present invention A method for producing a non-human animal model of an IgG4-related disease comprising: The present invention relates to a method comprising at least one of an anti-laminin antibody administration step of administering an antibody that immunologically reacts with laminin to a non-human animal, and a laminin immunization step of immunizing a non-human animal with laminin as an antigen.
  • laminin, antibody, etc. in the embodiment including the anti-laminin antibody administration step or laminin immunization step of the method for producing a non-human model animal of the present invention include the anti-laminin antibody detection step of the test method of the present invention. This is the same as in the embodiment.
  • Experiment 1 and Experiment 2 described in this specification it has been confirmed that when a serum obtained from an IgG4-related disease patient or an antibody purified from the serum is administered to the mouse, the mouse develops an IgG4-related disease.
  • serum obtained from a patient with an IgG4-related disease contains an antibody that immunologically reacts with laminin. From this, it can be said that a non-human model animal of an IgG4-related disease can be produced by an anti-laminin antibody administration step in which an antibody that immunologically reacts with laminin is administered to the non-human animal.
  • non-human animals in the anti-laminin antibody administration step include mice such as BALB / c mice and B6 mice, and other non-human animals.
  • the administration route of the antibody that immunologically reacts with laminin is not particularly limited, and examples thereof include subcutaneous administration, intraperitoneal administration, and intravenous administration.
  • An antibody that immunologically reacts with laminin need not be administered to a non-human animal as a purified antibody, and may be administered to a non-human animal as serum containing the antibody, for example.
  • the antibody that immunologically reacts with laminin is selected so that it can immunologically react with laminin of the non-human animal to be administered.
  • a human-derived anti-human laminin antibody can immunologically react with laminin possessed by mice, and can cause lesions of IgG4-related diseases in mice.
  • non-human animals in the laminin immunization process include mice such as BALB / c mice and B6 mice, and other non-human animals.
  • the method in the laminin immunization process is not particularly limited.
  • a non-human animal can be immunized by administering laminin together with a suitable adjuvant such as complete Freund's adjuvant to the non-human animal by a route such as subcutaneous administration, intravenous administration, or intraperitoneal administration.
  • laminin to be administered as an antigen is selected so that an antibody against it can immunologically respond to laminin possessed by the non-human animal to be immunized by autoimmunity.
  • human laminin-511 can react immunologically with mouse antibodies against mouse laminin, and can cause lesions of IgG4-related diseases in mice. ⁇ 8.
  • a method for producing a non-human animal model of an IgG4-related disease comprising: The present invention relates to a method comprising at least one of an anti-integrin antibody administration step of administering an antibody that immunologically reacts with an integrin to a non-human animal, and an integrin immunization step of immunizing the non-human animal with an integrin as an antigen.
  • the specific form of integrin, antibody, etc. includes the anti-integrin antibody detection step of the test method of the present invention. This is the same as in the embodiment.
  • non-human animals in the anti-integrin antibody administration step include mice such as BALB / c mice and B6 mice, and other non-human animals.
  • the administration route of the antibody that immunologically reacts with the integrin in the anti-integrin antibody administration step is not particularly limited, and examples thereof include subcutaneous administration, intraperitoneal administration, and intravenous administration.
  • the antibody that immunologically reacts with the integrin need not be administered to a non-human animal as a purified antibody, and may be administered to a non-human animal as serum containing the antibody, for example.
  • the antibody that immunologically reacts with the integrin is selected so that it can react immunologically with the integrin of the non-human animal to be administered.
  • a human-derived anti-human integrin antibody (particularly, a human-derived anti-human integrin ⁇ 6 ⁇ 1 antibody) can immunologically react with an integrin possessed by a mouse, and can cause a lesion of an IgG4-related disease in the mouse.
  • non-human animals in the integrin immunization step include mice such as BALB / c mice and B6 mice, and other non-human animals.
  • the method in the integrin immunization process is not particularly limited.
  • a non-human animal can be immunized by administering the integrin together with a suitable adjuvant such as complete Freund's adjuvant to the non-human animal by a route such as subcutaneous administration, intravenous administration, or intraperitoneal administration.
  • the integrin to be administered as an antigen is selected so that the antibody against it can react immunologically with the integrin of the non-human animal to be immunized by autoimmunity.
  • human integrins can react immunologically with mouse integrins by autoimmunity with mouse antibodies against them, and can cause lesions of IgG4-related diseases in mice.
  • Experiment 1 IgG administration of IgG4-related disease patients to mice>
  • IgG purified from the serum of an IgG4-related disease patient was administered to healthy newborn mice, and the effect on the pancreas after administration was observed. As a result, it was revealed that IgG purified from the serum of patients with IgG4-related diseases showed pathogenicity to the pancreas.
  • Serum serum samples were obtained from 10 patients diagnosed with IgG4-related disease.
  • the concentration of purified IgG was measured using a Human IgG EIA Kit (MK136, manufactured by Takara).
  • the purity of the IgG fraction was determined using Human IgA ELISA Kit (E88-102, manufactured by Ethyl Laboratories), Human IgM ELISA Kit (manufactured by E88-100, manufactured by Ethyl Laboratores), Human IgE ELISA 108-L (manufactured by Human IgE ELISA-Lit).
  • the contamination of IgA, IgM, IgE and protein was determined and confirmed using Coomassie brilliant blue staining, respectively.
  • IgG obtained from a serum sample of an IgG4-related disease patient was designated as “patient IgG”.
  • the IgG obtained from the control serum sample was designated as “control IgG”.
  • Animal test Male newborns of BALB / c mice were used as mice.
  • IgG prepared from each serum sample was administered subcutaneously to mice. 10-20 mg of human IgG was administered to 1 g of mouse body weight. 1.4. Results and discussion 1 (FIGS. 1A to 1C) Twelve hours after subcutaneous administration of patient IgG or control IgG, mouse pancreas tissue sections were collected, stained by H & E (hematoxylin & eosin) staining and immunohistochemical staining, and observed. In immunohistochemical staining, Gr1-positive cells were stained using an antibody against bone marrow cell differentiation antigen Gr1 as a primary antibody.
  • the human IgG concentration in the mouse serum was measured 12 hours after subcutaneous administration.
  • FIG. 1A shows the results of H & E staining of pancreas 12 hours after IgG administration (upper row) and Gr1 staining (lower row) of control IgG-treated mice (left column) and patient IgG-treated mice (right column).
  • the scale bar in the observed image shows 50 ⁇ m in the upper stage and 20 ⁇ m in the lower stage.
  • FIG. 1B shows the correlation between the dose of control IgG (triangle) and patient IgG (circle) and the edema area.
  • the cutoff value (21.1%) in the figure is a value obtained by adding a value twice the standard deviation (SD) to the average value of the edema area in the control IgG or PBS-administered mice.
  • FIG. 1C shows the histopathological grade of pancreatic necrosis or hemorrhage 12 hours after IgG administration and the number of Gr1-positive cells in control IgG-treated mice (left) and patient IgG-treated mice (right) ( * p ⁇ 0.05, ** p ⁇ 0.005: according to Paired Student's test).
  • FIG. 1B there was no significant difference in serum human IgG concentrations between patient IgG-administered mice and control IgG-administered mice (19.4 mg / ml and 21.4 mg / ml).
  • pancreas was damaged by administration of patient IgG to mice, and the damage was maximized 12 hours after administration as shown in FIG. 1A.
  • Administration of control IgG to mice did not cause pancreatic damage 12 hours after administration, as shown in FIG. 1A.
  • mouse pancreas tissue sections were collected, stained by immunohistochemical staining, and observed.
  • immunohistochemical staining anti-human IgG antibody, anti-human IgG1 antibody or anti-human IgG4 antibody was used as a primary antibody, and human IgG, human IgG1 or human IgG4 was stained by a conventional method.
  • mouse pancreas tissue section was stained by immunofluorescence staining and observed with a fluorescence microscope.
  • immunofluorescence staining human IgG, amylase or collagen IV was stained by a conventional method using a rabbit anti-human IgG antibody, rabbit anti-amylase antibody or rabbit anti-collagen IV antibody conjugated with fluorescein isocyanate.
  • FIG. 2A shows immunization of IgG (upper), IgG1 (middle), and IgG4 (lower) in pancreas 12 hours after IgG administration in control IgG-treated mice (left column) and patient IgG-treated mice (right column). The results of histochemical staining are shown. The scale bar in the observed image indicates 20 ⁇ m.
  • FIG. 2B shows the results of immunofluorescence staining of pancreatic tissue of control IgG-administered mice and patient IgG-administered mice.
  • the scale bar in the observed image indicates 20 ⁇ m.
  • FIG. 2B shows an observed image of immunofluorescent-stained human IgG in the pancreatic tissue of a control IgG-treated mouse. Human IgG was not observed in the mouse pancreas.
  • the second from the left in the upper part of FIG. 2B shows an observation image of human IgG stained with immunofluorescence in the pancreatic tissue of a patient IgG-administered mouse.
  • the third from the left in the upper part of FIG. 2B shows an observation image of amylase stained with immunofluorescence in the same observation region as that in the second from the left in the upper part of FIG.
  • the right end of the upper stage of FIG. 2B is an image obtained by superimposing the above observed image of human IgG and the above observed image of amylase.
  • the first from the left in the lower part of FIG. 2B shows an observation image of human IgG stained with immunofluorescence in the pancreatic tissue of a patient IgG-administered mouse.
  • the second from the left in the lower part of FIG. 2B shows an observation image of collagen IV stained with immunofluorescence in the same observation area as the first from the left in the lower part of FIG. .
  • the lower right end of FIG. 2B is an image obtained by superimposing the observed image of the above human IgG and the observed image of the above collagen IV.
  • IgG and IgG4 were observed to accumulate in the pancreatic acinar base and pancreatic tissue lobule gap in all patient IgG-treated mice, whereas in control IgG-treated mice pancreas IgG and IgG4 were not observed.
  • IgG1 was observed in 6 out of 10 mice administered with patient IgG, but IgG1 was not observed in mice administered with control IgG.
  • IgG subclasses IgG1 and IgG4 were purified from sera of patients with IgG4-related diseases and administered to healthy newborn mice, and the effect on the pancreas after administration was observed.
  • IgG1 and IgG4 purified from the serum of patients with IgG4-related diseases show pathogenicity to the pancreas, IgG1 pathogenicity is greater than that of IgG4, and administered in combination with IgG1 and IgG4 Then, it was confirmed that the pathogenicity of IgG1 was suppressed.
  • IgG1 and IgG4 were purified from the serum samples of the patients with IgG4-related diseases and the control serum samples obtained.
  • Capture Select IgG1 (Hu) affinity matrix (191303005, manufactured by Invitrogen) and Capture Select IgG4 (Hu) affinity matrix (manufactured by Invitrogen, manufactured by Invitrogen) were used.
  • the fraction of the eluted subclass (IgG1 or IgG4) was concentrated by ultrafiltration using Amicon Ultra (UFC805024, manufactured by Millipore) while thoroughly washing with PBS (pH 7.2).
  • the concentration of the purified IgG subclass was determined by using the Human IgG1 Platinum ELISA (BMS2092, eBioscience), Human IgG2 Platinum ELISA (BMS2093, eBioscience), Human IgG3 Platinum ELISA (BMS2094, eBioScientium BMS2094, eBioSc4).
  • Product was used and quantified according to the product instructions. The purity of the subclass was confirmed by determining the contamination of IgA, IgM, IgE, other IgG subclasses and proteins by the same procedure as described above.
  • IgG1 and IgG4 obtained from serum samples of patients with IgG4-related diseases were designated as “patient IgG1” and “patient IgG4”, respectively.
  • IgG1 and IgG4 obtained from the control serum samples were designated as “control IgG1” and “control IgG4”, respectively.
  • IgG1 and IgG4 prepared from each serum sample was administered subcutaneously to mice.
  • IgG1 is administered in an amount of 10 mg / mouse body weight of 1 g.
  • IgG4 is administered in an amount of 1 mg / mouse body weight of 1 g
  • IgG1 and IgG4 Were administered in an amount of 1 mg / mouse weight of 1 g of IgG4.
  • Control 1 in the upper part of FIG. 3A shows pancreatic H & E 12 hours after administration of mice administered with one or both of control IgG1 and control IgG4 obtained from a specific one of the 10 controls (control 1). It is an observation image of dyeing
  • “Patient 9” in the middle and bottom of FIG. 3A shows 12 hours of administration of a mouse administered with one or both of patient IgG1 and patient IgG4 obtained from one specific patient (patient 9) among 10 IgG4-related disease patients. It is the observation image (lower stage) of H & E staining of the pancreas after, and the observation image (lower stage) of immunohistochemical staining of Gr1. The scale bar in the observed image indicates 20 ⁇ m.
  • FIG. 3B shows pancreatic necrosis or 12 hours after administration of mice that received one or both of control IgG1 and control IgG4 (left) and mice that received one or both of patient IgG1 and patient IgG4 (right).
  • the histopathological grade of bleeding and the number of Gr1 positive cells are shown ( * p ⁇ 0.05: according to Paired Student's test).
  • both patient IgG1 and patient IgG4 are pathogenic to the pancreas, the pathogenicity of patient IgG1 is greater than the pathogenicity of patient IgG4, and patient IgG1 when administered together with patient IgG1 and patient IgG4 It was confirmed that the pathogenicity of was suppressed.
  • FIG. 3C shows pancreatic human IgG1 or human 12 hours after administration of mice administered with one or both of patient IgG1 and patient IgG4 from one particular patient (patient 1) out of 10 IgG4-related diseases It is an observation image of immunohistochemical staining of IgG4.
  • the scale bar in the observed image indicates 20 ⁇ m.
  • Human IgG1 staining intensity of pancreas in mice administered with both patient IgG1 and patient IgG4 (upper right of FIG. 3C) is greater than human IgG1 staining intensity of pancreas in mice administered only with patient IgG1 (upper left of FIG. 3C) It was confirmed to be small.
  • the human IgG4 staining intensity of the pancreas in the mouse administered with both patient IgG1 and patient IgG4 is the human IgG4 staining intensity of the pancreas in the mouse administered only with patient IgG4 (upper in FIG. 3C) It was comparable.
  • mice administered with one or both of patient IgG1 and patient IgG4 edema formation, acinar necrosis, hemorrhage and infiltration of Gr1-positive polymorphonuclear leukocytes were observed in the pancreas.
  • no abnormality was observed in the pancreas in mice administered with one or both of control IgG1 and control IgG4.
  • IgG1 and IgG4 are pathogenic, and IgG1 is more pathogenic than IgG4.
  • the pathogenicity of IgG1 was markedly suppressed when IgG4 was administered simultaneously, suggesting that IgG4 inhibits the action of IgG1 (FIG. 3B).
  • the amount of IgG1 staining in mice administered only with IgG1 is significantly greater than when IgG1 and IgG4 are administered in combination (FIG. 3C).
  • the amount of IgG4 staining in mice administered with IgG4 alone was similar to that obtained when IgG1 and IgG4 were administered in combination (FIG. 3C).
  • FIG. 4A shows an observation image obtained by H & E staining of a pancreatic tissue section of an AIP patient collected from a boundary portion between a site with AIP lesion (left side) and a site without lesion (right side).
  • the upper right of FIG. 4A shows an observation image of immunohistochemical staining for IgG4 of the same pancreatic tissue section.
  • the scale bar indicates 200 ⁇ m.
  • the lower left of FIG. 4A is an enlarged image of the lesioned acinar portion in the upper right image of FIG. 4A.
  • the lower part of FIG. 4A is an observation image obtained by enlarging the lesioned leaflet gap portion in the upper right part of FIG. 4A.
  • the lower right side of FIG. 4A is an observation image obtained by enlarging a normal acinar portion without a lesion in the upper right image of FIG. 4A.
  • the scale bar indicates 20 ⁇ m.
  • IgG4 positive plasma cells were stained, and IgG4 was linearly stained at the base of the acinus and the lobule space. On the other hand, such staining was not observed in the pancreatic tissue without lesion.
  • FIG. 4B shows an observation image obtained by immunofluorescent staining of an acinus having a lesion in the pancreas of an AIP patient.
  • the scale bar indicates 20 ⁇ m.
  • FIG. 4B upper left, shows an observed image of IgG4 stained with immunofluorescence in the acinus of the AIP patient with a lesion in the pancreas.
  • the upper part of FIG. 4B shows an observation image of amylase stained with immunofluorescence in the same observation area as the upper left part of FIG. 4B.
  • the upper right of FIG. 4B is an image obtained by superimposing the observed image of IgG4 on the upper left of FIG. 4B and the observed image of amylase in the upper part of FIG. 4B. From this result, it can be seen that IgG4 does not coexist with amylase and exists at different sites.
  • FIG. 4B lower left, shows an observed image of immunofluorescent-stained IgG4 in an acinus having a lesion in the pancreas of an AIP patient.
  • the lower part of FIG. 4B shows an observation image of collagen IV stained with immunofluorescence in the same observation area as the lower left part of FIG. 4B.
  • the lower right of FIG. 4B is an image obtained by superimposing the observed image of IgG4 on the lower left of FIG. 4B and the observed image of collagen IV in the lower part of FIG. 4B.
  • a portion where red IgG4 and green collagen IV overlap is shown in yellow. From this result, it can be seen that IgG4 is present at the base of the acinar together with collagen IV.
  • FIG. 4C shows an observation image obtained by immunofluorescent staining of a lobular space with a lesion in the pancreas of an AIP patient.
  • the scale bar indicates 20 ⁇ m.
  • FIG. 4C left shows an observed image of IgG4 stained with immunofluorescence in the lobular space with lesions in the pancreas of an AIP patient.
  • FIG. 4C shows an observation image of collagen IV stained with immunofluorescence in the same observation region as the left of FIG. 4C.
  • the right side of FIG. 4C is an image obtained by superimposing the observed image of IgG4 on the left side of FIG. 4C and the observed image of collagen IV in FIG. 4C.
  • On the right side of FIG. 4C a portion where red IgG4 and green collagen IV overlap is shown in yellow. From this result, it can be seen that IgG4 exists in the extracellular matrix expressing collagen IV.
  • the present inventors speculated that the antibody contained in the serum of the AIP patient recognizes an extracellular matrix protein expressed in the pancreas as an antigen.
  • the inventors of the present invention individually fixed an extracellular matrix protein expressed in the pancreas known from the literature as an antigen candidate on a solid phase, and in the serum of an AIP patient, Whether or not an antibody that binds to an extracellular matrix protein was contained was confirmed by ELISA (Enzyme-Linked Immunosorbent Assay) method.
  • Human laminin-511-E8 (892012, manufactured by Nippi Co., Ltd.) (Laminin-511 active fragment) Human collagen IV (ab7536, abcam) Human fibronectin GFP (green fluorescent protein) (MB-0752, VECTOR LABORATORIES) Human Notch1 (ab68580, abcam) Human Annexin A2 (ab93005, abcam) Human SDF2L1 (TP313102, OriGene Technologies) Human Bile salt-activated lipidase (ab167963, abcam) 4.2. Serum samples Serum samples were obtained from 9 patients diagnosed with AIP.
  • ELISA coating buffer (ELISA Coating Buffer), ELISA wash solution (ELISA Wash Solution), ELISA blocking buffer (ELISA Blocking Buffer), and conjugate diluent (Conjugate Diluent) were all prepared according to the instructions of the kit.
  • HRP-conjugated secondary antibody (1) Secondary antibody conjugated with HRP (horseradish peroxidase) (abcam6759, rabbit anti-human IgG H & L (HRP), polyclonal) is conjugated diluent of the kit (Conjugate Diluent) Diluted 1: 2000.
  • Enzyme substrate reaction (1) A TMB (3,3 ′, 5,5′-tetramethylbenzidine) solution was prepared according to the manufacturer's recommended conditions.
  • FIG. 5A anti-human laminin-511 active fragment antibody
  • FIG. 5B anti-human collagen IV antibody
  • FIG. 5C anti-human fibronectin antibody
  • FIG. 5D anti-GFP antibody
  • n 40 for the AIP patient serum sample and for the control serum sample.
  • the vertical axis represents the relative absorbance at 450 nm, which is proportional to the serum concentration of the antibody against each antigen candidate.
  • the cut-off value is a value obtained by adding a value twice the standard deviation (SD) to the average value of the serum concentration of the antibody against each antigen candidate in the control serum sample. When the serum concentration of the antibody against the antigen candidate is higher than the cutoff value, it can be determined that the antibody against the antigen candidate is positive.
  • anti-human laminin-511 antibody was positive in 20 of 40 cases in AIP patient serum, whereas no positive case was found in 40 cases in control serum. This confirms that AIP can be detected using the serum concentration of anti-human laminin-511 antibody as an index. Further, in one of the AIP patient sera positive for anti-human laminin-511 antibody, the blood IgG concentration and blood IgG4 concentration were normal values with no substantial difference from the control serum. The method of discriminating AIP using the serum concentration of ⁇ 511 antibody as an index may be able to discriminate AIP with higher sensitivity than the method using blood IgG concentration and blood IgG4 concentration as an index.
  • mice were treated in the same procedure and schedule except that 120 ⁇ g of ovalbumin (OVA) emulsified with CFA was used instead of the emulsion of laminin-511-E8.
  • OVA ovalbumin
  • Sample sera are blood serum (pre-immune serum) collected from mice prior to the first dose and blood serum collected after sacrifice. Sample serum was stored at ⁇ 80 ° C. until analysis. Tissue sections of mouse pancreas were collected 28 days after the third immunization and stained with H & E staining for observation.
  • FIG. 6 shows an observation image of a tissue section of the pancreas stained by H & E staining.
  • FIG. 6 left is an observation image of a control group
  • FIG. 6 right is an observation image of a test group immunized with a human laminin-511 active fragment (i511 laminin).
  • i511 laminin a human laminin-511 active fragment
  • Human recombinant laminin 521 (BLA-LN521-02, Bio Lamina) was used as human laminin-521.
  • IgG4-related kidney disease patient IgG The serum concentration of anti-human laminin-521 antibody may be effective as an indicator of discrimination for IgG4-related renal diseases.
  • Experiment 7 Administration of IgG4 Related Kidney Disease Patient IgG to Mice> IgG was prepared from the serum of an IgG4-related renal disease patient who was confirmed to be positive for anti-human laminin-521 antibody in Experiment 6 by the same procedure as in Experiment 1. This IgG was designated as “IgG4-related kidney disease patient IgG”.
  • IgG was prepared from the serum of an IgG4-related disease patient who has no kidney disease by the same procedure as in Experiment 1. This IgG was designated as “IgG4 related disease patient IgG without renal disease”.
  • IgG4-related kidney disease patient IgG, control IgG, and IgG4-related disease patient IgG without kidney disease were each subcutaneously administered to male newborns of BALB / c mice in the same procedure as in Experiment 1.
  • mice kidneys were collected in the same manner as in Experiment 1, and human IgG4 was stained and observed by immunohistochemical staining.
  • FIG. 8 shows the observation image of the kidney of the mouse subcutaneously administered with IgG4 related kidney disease patient IgG on the left side of FIG. 8, and shows the human IgG4 staining observation image of the kidney of the mouse subcutaneously administered with the control IgG on the right side of FIG.
  • the upper row shows the human IgG4 staining observation image of the whole kidney
  • the lower row shows the human IgG4 staining observation image of the filamentous body.
  • Pierson syndrome is caused by a genetic abnormality of laminin ⁇ 2 chain.
  • Pierson's syndrome includes renal failure (mesangial proliferating filamentous nephritis), neuropathy (muscular weakness), and miosis.
  • Anti-human laminin-521 antibody-positive IgG4-related kidney disease patients who have obtained the above IgG4-related kidney disease patient IgG have been confirmed to have mesangial proliferative fibronephritis by renal biopsy, and muscle weakness has also been confirmed. Therefore, Pierson's syndrome matches symptoms and pathology.
  • this IgG4-related renal disease patient has an autoantibody against laminin ⁇ 2 chain, which is presumed to cause symptoms of IgG4-related renal disease.
  • Experiment 8 Measurement of anti-laminin 511-E8 autoantibodies in serum of AIP patients> 8.1. Samples Serum samples from 51 in-treatment AIP patients who met the diagnostic criteria for IgG4-RD 2011 or the diagnostic criteria for AIP were obtained. Control serum samples were obtained from 25 healthy individuals and 86 disease patients (49 histologically confirmed cancer patients and 39 non-cancer disease patients). Serum samples from 10 of 51 AIP patients and serum samples from 10 of 112 controls were used as training groups, and the rest were used as validation groups.
  • Human laminin-511-E8 was diluted in the ELISA coating buffer of the kit to obtain a 2 ⁇ g / ml concentration solution, and this solution was added to the microtiter plate of the kit so as to be 100 ⁇ l per well. Incubate for time to coat. The plate was washed five times with 0.05% Tween 20-containing Tris buffered saline (washing solution), then coated with 1% bovine serum albumin-containing Tris buffered saline, and 100 ⁇ L of 1:20 diluted serum. And incubated at room temperature for 30 minutes.
  • the plate After washing 5 times with the washing solution, the plate was incubated with 100 ⁇ L of goat anti-human IgG antibody conjugated with HRP (horseradish peroxidase) (1: 4000; ab6759, Abcam) for 1 hour at room temperature. . After washing with the washing solution three times, the plate was incubated with TMB (3,3 ′, 5,5′-tetramethylbenzidine) for 3 minutes, and the absorbance at 450 nm was measured to quantify the amount of bound substance. Control wells not coated with human laminin-511-E8 were used as negative controls in the ELISA for each serum sample.
  • HRP horseradish peroxidase
  • the cut-off value (optical density unit [OD]) is a value obtained by adding three times the standard deviation (SD) to the mean value of anti-human laminin-511-E8 IgG antibody level in the serum sample from the control. is there. Serum samples from 6 of 10 AIP patients were positive for IgG antibodies to human laminin-511-E8, whereas all 10 controls had IgG antibodies to human laminin-511-E8. Negative.
  • FIG. 9B shows the measurement results of anti-human laminin-511-E8 IgG antibody levels of serum samples from 41 AIP patients in the validation group and 102 controls (20 healthy subjects, 82 disease patients other than AIP). Shown in The cut-off value (optical density unit [OD]) is a value obtained by adding 3 times the standard deviation (SD) to the mean value of anti-human laminin-511-E8 IgG antibody level in the serum sample from the control. is there. Serum samples from 20 of 41 AIP patients had anti-human laminin-511-E8 IgG antibody (positive), whereas only 2 out of 102 controls Anti-human laminin-511-E8 IgG antibody was positive.
  • SD standard deviation
  • the IgG antibody positive against human laminin-511-E8 was 26/51 (51.0%) in the serum of AIP patients, whereas it was 2 in the control serum. / 112 (1.8%) (P ⁇ 0.001).
  • integrin ⁇ 6 ⁇ As the integrin ⁇ 6 ⁇ 1, recombinant human integrin ⁇ 6 ⁇ 1 (R & D Systems, Minnesota, USA, product number 7809-A6) was used.
  • FIG. 10 shows the measurement results of the anti-integrin ⁇ 3 ⁇ 1 IgG antibody level, respectively.
  • the cut-off value (optical density unit [OD]) is the mean value of anti-integrin ⁇ 6 ⁇ 1 IgG antibody level or anti-integrin ⁇ 3 ⁇ 1 IgG antibody level in the serum sample from the control, which is 3 times the standard deviation (SD). It is the added value.
  • AIP patients were divided into 26 anti-laminin 511-E8 antibody positive AIP patients (anti-integrin ⁇ 6 ⁇ 1 antibody negative) and 4 anti-integrin ⁇ 6 ⁇ 1 antibody positive AIP patients (anti-laminin 511-E8 antibody negative).
  • the 21 anti-laminin 511-E8 antibody negative anti-integrin ⁇ 6 ⁇ 1 antibody negative AIP patients were classified, and the clinical features of each group of AIP patients are summarized in Table 5 below. There were no subjects who were positive for both anti-laminin 511-E8 antibody and anti-integrin ⁇ 6 ⁇ 1 antibody.
  • pancreatic images Comparison of pancreatic images>
  • the upper part of FIG. 12 is a pancreatic image by contrast-enhanced CT of an anti-laminin 511-E8 antibody positive AIP patient.
  • the lower part of FIG. 12 is a pancreatic image obtained by contrast CT of an anti-integrin ⁇ 6 ⁇ 1 antibody-positive AIP patient.
  • the arrow in each image indicates the lesion site.
  • FIG. 13 shows the serum anti-laminin 511-E8 antibody levels of five anti-laminin 511-E8 antibody positive AIP patients before and after steroid treatment.
  • Steroid treatment reduced the anti-laminin 511-E8 antibody titer in the serum of each AIP patient below the cut-off value.
  • pancreatic imaging findings also improved with the reduction of the anti-laminin 511-E8 antibody titer in the serum of each AIP patient. This confirms that anti-laminin 511-E8 antibody in serum is also useful as an indicator of the effect of AIP treatment.

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Abstract

The present invention addresses the problem of finding a pathogenic substance causing an IgG4-related disease and providing a method for testing for an IgG4-related disease and others using the pathogenic substance. The present invention provides a method for testing for an IgG4-related disease, said method comprising an anti-laminin antibody detection step of detecting, as an indicator for an IgG4-related disease, an antibody capable of immunologically reacting with laminin in a sample and/or an anti-integrin antibody detection step of detecting, as an indicator for an IgG4-related disease, an antibody capable of immunologically reacting with integrin in the sample. The present invention also provides: a test reagent for use in the testing for an IgG4-related disease, which contains laminin; and a test reagent for use in the testing for an IgG4-related disease, which contains integrin.

Description

ＩｇＧ４関連疾患の検査方法Method for testing IgG4-related disease
　本発明は、ＩｇＧ４関連疾患の検査方法、ＩｇＧ４関連疾患を検査するための検査試薬、ＩｇＧ４関連疾患に対する治療の効果を評価する方法、及び、ＩｇＧ４関連疾患の治療薬の候補物質をスクリーニングする方法に関する。 The present invention relates to a test method for IgG4-related diseases, a test reagent for testing IgG4-related diseases, a method for evaluating the effect of treatment on IgG4-related diseases, and a method for screening candidate substances for therapeutic agents for IgG4-related diseases. .
　ＩｇＧ４関連疾患とは、血清ＩｇＧ４高値とＩｇＧ４陽性形質細胞の組織浸潤または腫瘤形成を特徴とする疾患群である。 IgG4-related diseases are disease groups characterized by tissue infiltration or tumor formation of high serum IgG4 levels and IgG4-positive plasma cells.
　ＩｇＧ４関連疾患は比較的新しい疾患概念であり、これまでのところ原因は不明である。罹患臓器としては膵臓、胆管、涙腺・唾液腺、中枢神経系、甲状腺、肺、肝臓、消化管、腎臓、前立腺、後腹膜、動脈、リンパ節、皮膚、乳腺等が知られている。病変が複数臓器におよび全身疾患としての特徴を有することが多いが、単一臓器病変の場合もある。 IgG4-related disease is a relatively new disease concept, and the cause is unknown so far. As affected organs, pancreas, bile duct, lacrimal gland, salivary gland, central nervous system, thyroid gland, lung, liver, gastrointestinal tract, kidney, prostate, retroperitoneum, artery, lymph node, skin, mammary gland and the like are known. Lesions often have features in multiple organs and as systemic disease, but may be single organ lesions.
　自己免疫性膵炎（ＡＩＰ）、ＩｇＧ４関連腎疾患、ＩｇＧ４関連涙腺唾液腺炎等がＩｇＧ４関連疾患に分類される疾患として知られている。 Autoimmune pancreatitis (AIP), IgG4-related kidney disease, IgG4-related lacrimal salivary glanditis and the like are known as diseases classified as IgG4-related diseases.
　ＩｇＧ４関連疾患の原因は従来知られておらず、ＩｇＧ４関連疾患の指標となるマーカー物質も知られていない。このため従来ＩｇＧ４関連疾患であると診断することは容易ではなかった。例えば、ＡＩＰは膵臓での組織浸潤を特徴とするが、組織の観察像からは膵癌との判別が難しく、ＡＩＰが誤って膵癌と診断される可能性もあった。 The cause of IgG4-related diseases has not been known so far, and a marker substance that serves as an indicator of IgG4-related diseases has not been known. For this reason, it was not easy to diagnose it as an IgG4-related disease. For example, although AIP is characterized by tissue infiltration in the pancreas, it is difficult to distinguish it from pancreatic cancer from the observed image of the tissue, and AIP may be erroneously diagnosed as pancreatic cancer.
特開２００９－２９４０４０号公報JP 2009-294040 A 特開２００８－２７５５２７号公報JP 2008-275527 A
　これまでに自己免疫性膵炎の検査方法として、ＨＳＰ１０と免疫学的に反応する抗体を検出する方法（特許文献１）や、アミラーゼα２－Ａと免疫学的に反応する抗体を検出する方法（特許文献２）が知られている。しかしながら、これらの方法は、自己免疫性膵炎の検査方法として必ずしも満足できるものではない。 To date, methods for detecting autoimmune pancreatitis include a method for detecting an antibody that immunologically reacts with HSP10 (Patent Document 1) and a method for detecting an antibody that immunologically reacts with amylase α2-A (patent 1). Document 2) is known. However, these methods are not always satisfactory as a test method for autoimmune pancreatitis.
　ＩｇＧ４関連疾患の病原物質が特定できれば、病原物質を指標としてＩｇＧ４関連疾患を検査すること、病原物質を用いてＩｇＧ４関連疾患のモデル動物を作製すること、病原物質を標的として新たな治療薬を開発すること、等が可能になる。しかし、これまでのところＩｇＧ４関連疾患の病原物質は解明されていない。 If the pathogen of IgG4-related disease can be identified, testing for IgG4-related disease using the pathogen as an index, creating a model animal of IgG4-related disease using the pathogen, and developing a new therapeutic drug targeting the pathogen And so on. However, so far, pathogenic substances for IgG4-related diseases have not been elucidated.
　ＩｇＧ４関連疾患の病原物質を明らかにし、それを利用して、ＩｇＧ４関連疾患の検査方法、ＩｇＧ４関連疾患を検査するための検査試薬、ＩｇＧ４関連疾患に対する治療の効果を評価する方法、ＩｇＧ４関連疾患の治療薬の候補物質をスクリーニングする方法として有効な手段を提供することが求められている。 Clarifying the pathogens of IgG4-related diseases and using them, methods for testing IgG4-related diseases, test reagents for testing IgG4-related diseases, methods for evaluating the effects of treatment on IgG4-related diseases, IgG4-related diseases There is a need to provide an effective means for screening candidate substances for therapeutic drugs.
　本発明者らは驚くべきことに、ＩｇＧ４関連疾患の病原物質が、ラミニンに対する自己抗体であること、及び、血清中における抗ラミニン抗体及び抗インテグリン抗体がＩｇＧ４関連疾患の指標となることを解明し、この知見に基づいて以下の発明を完成するに至った。
（１）ＩｇＧ４関連疾患の検査方法であって、
　ＩｇＧ４関連疾患の指標として、検体中の、ラミニンと免疫学的に反応する抗体を検出する抗ラミニン抗体検出工程、及び／又は
　ＩｇＧ４関連疾患の指標として、検体中の、インテグリンと免疫学的に反応する抗体を検出する抗インテグリン抗体検出工程
を含む方法。
（２）前記ＩｇＧ４関連疾患が、自己免疫性膵炎であり、
　抗ラミニン抗体検出工程における前記抗体が、ラミニン－５１１と免疫学的に反応する抗体である、（１）に記載の方法。
（３）前記ＩｇＧ４関連疾患が、自己免疫性膵炎であり、
　抗インテグリン抗体検出工程における前記抗体が、インテグリンα６β１と免疫学的に反応する抗体である、（１）又は（２）に記載の方法。
（４）前記ＩｇＧ４関連疾患が、ＩｇＧ４関連腎疾患であり、
　抗ラミニン抗体検出工程における前記抗体が、ラミニン－５２１と免疫学的に反応する抗体である、（１）に記載の方法。
（５）前記抗ラミニン抗体検出工程が、ラミニンを抗原として用いて前記抗体を検出することを含む、（１）～（４）のいずれかに記載の方法。
（６）前記抗インテグリン抗体検出工程が、インテグリンを抗原として用いて前記抗体を検出することを含む、（１）～（５）のいずれかに記載の方法。
（７）前記検体が血液試料である、（１）～（６）のいずれかに記載の方法。
（７’）自己免疫性膵炎の検査方法であって、
　自己免疫性膵炎の指標として、検体の血液試料中の、ラミニン－５１１と免疫学的に反応する抗体を検出する抗ラミニン抗体検出工程、及び
　自己免疫性膵炎の指標として、検体の血液試料中の、インテグリンα６β１と免疫学的に反応する抗体を検出する抗インテグリン抗体検出工程
　を含む方法。
（８）ラミニンを含む、ＩｇＧ４関連疾患を検査するための検査試薬。
（９）前記ＩｇＧ４関連疾患が、自己免疫性膵炎であり、
　前記ラミニンが、ラミニン－５１１である、（８）に記載の検査試薬。
（１０）前記ＩｇＧ４関連疾患が、ＩｇＧ４関連腎疾患であり、
　前記ラミニンが、ラミニン－５２１である、（８）に記載の検査試薬。
（１１）前記ラミニンを、固相に固定された形態で含む、（８）～（１０）のいずれかに記載の検査試薬。
（１２）インテグリンを含む、ＩｇＧ４関連疾患を検査するための検査試薬。
（１３）前記ＩｇＧ４関連疾患が、自己免疫性膵炎であり、
　前記インテグリンが、インテグリンα６β１である、（１２）に記載の検査試薬。
（１４）前記インテグリンを、固相に固定された形態で含む、（１２）又は（１３）に記載の検査試薬。
（１４’）ラミニン－５１１とインテグリンα６β１とを固相に固定された形態で含む、自己免疫性膵炎の検査試薬。
（１５）ＩｇＧ４関連疾患に対する治療の効果を評価する方法であって、
　ＩｇＧ４関連疾患に対する治療が施された被検動物から得た検体中の、ラミニンと免疫学的に反応する抗体を検出する抗ラミニン抗体検出工程、及び／又は、
　ＩｇＧ４関連疾患に対する治療が施された被検動物から得た検体中の、インテグリンと免疫学的に反応する抗体を検出する抗インテグリン抗体検出工程
を含む方法。
（１６）ＩｇＧ４関連疾患の治療薬の候補物質をスクリーニングする方法であって、
　試験物質を作用させた動物から得た検体中の、ラミニンと免疫学的に反応する抗体を検出する抗ラミニン抗体検出工程と、
　前記試験物質を作用させたことにより前記検体中の前記抗体が減少した場合に、前記試験物質を、ＩｇＧ４関連疾患の治療薬の候補物質として選抜する選抜工程と
を含む方法。
（１７）ＩｇＧ４関連疾患の治療薬の候補物質をスクリーニングする方法であって、
　試験物質を作用させた動物から得た検体中の、インテグリンと免疫学的に反応する抗体を検出する抗インテグリン抗体検出工程と、
　前記試験物質を作用させたことにより前記検体中の前記抗体が減少した場合に、前記試験物質を、ＩｇＧ４関連疾患の治療薬の候補物質として選抜する選抜工程と
を含む方法。
（１８）ＩｇＧ４関連疾患の非ヒトモデル動物を作製する方法であって、
　非ヒト動物に、ラミニンと免疫学的に反応する抗体を投与する抗ラミニン抗体投与工程、及び
　非ヒト動物を、ラミニンを抗原として免疫するラミニン免疫工程
のうち少なくとも一方を含む方法。
（１９）ＩｇＧ４関連疾患の非ヒトモデル動物を作製する方法であって、
　非ヒト動物に、インテグリンと免疫学的に反応する抗体を投与する抗インテグリン抗体投与工程、及び
　非ヒト動物を、インテグリンを抗原として免疫するインテグリン免疫工程
のうち少なくとも一方を含む方法。
（２０）ＩｇＧ４関連疾患の指標を取得する方法であって、
　検体中の、ラミニンと免疫学的に反応する抗体を検出する抗ラミニン抗体検出工程、及び／又は
　検体中の、インテグリンと免疫学的に反応する抗体を検出する抗インテグリン抗体検出工程
を含む方法。
（２１）前記ＩｇＧ４関連疾患が、自己免疫性膵炎であり、
　抗ラミニン抗体検出工程における前記抗体が、ラミニン－５１１と免疫学的に反応する抗体である、（２０）に記載の方法。
（２２）前記ＩｇＧ４関連疾患が、自己免疫性膵炎であり、
　抗インテグリン抗体検出工程における前記抗体が、インテグリンα６β１と免疫学的に反応する抗体である、（２０）又は（２１）に記載の方法。
（２３）前記ＩｇＧ４関連疾患が、ＩｇＧ４関連腎疾患であり、
　抗ラミニン抗体検出工程における前記抗体が、ラミニン－５２１と免疫学的に反応する抗体である、（２０）に記載の方法。
（２４）前記抗ラミニン抗体検出工程が、ラミニンを抗原として用いて前記抗体を検出することを含む、（２０）～（２３）のいずれかに記載の方法。
（２５）前記抗インテグリン抗体検出工程が、インテグリンを抗原として用いて前記抗体を検出することを含む、（２０）～（２４）のいずれかに記載の方法。
（２６）前記検体が、被検動物から分離された血液試料である、（２０）～（２５）のいずれかに記載の方法。
（２６’）自己免疫性膵炎の指標を取得する方法であって、
　検体中の、ラミニン－５１１と免疫学的に反応する抗体を検出する抗ラミニン抗体検出工程、及び
　検体中の、インテグリンα６β１と免疫学的に反応する抗体を検出する抗インテグリン抗体検出工程
を含む方法。
（２７）ＩｇＧ４関連疾患を検査するための検査試薬の製造における、ラミニンの使用。
（２８）前記ＩｇＧ４関連疾患が、自己免疫性膵炎であり、
　前記ラミニンが、ラミニン－５１１である、（２７）に記載の使用。
（２９）前記ＩｇＧ４関連疾患が、ＩｇＧ４関連腎疾患であり、
　前記ラミニンが、ラミニン－５２１である、（２７）に記載の使用。
（３０）前記ラミニンが、ラミニンが固定された固相の形態である、（２７）～（２９）のいずれかに記載の使用。
（３１）ＩｇＧ４関連疾患を検査するための検査試薬の製造における、インテグリンの使用。
（３２）前記ＩｇＧ４関連疾患が、自己免疫性膵炎であり、
　前記インテグリンが、インテグリンα６β１である、（３１）に記載の使用。
（３３）前記インテグリンが、インテグリンが固定された固相の形態である、（３１）又は（３２）に記載の使用。
（３３’）自己免疫性膵炎の検査試薬の製造における、固相に固定された形態の、ラミニン－５１１及びインテグリンα６β１の使用。
（３４）ＩｇＧ４関連疾患の検査に使用するためのラミニン。
（３５）前記ＩｇＧ４関連疾患が、自己免疫性膵炎であり、
　前記ラミニンが、ラミニン－５１１である、（３４）に記載のラミニン。
（３６）前記ＩｇＧ４関連疾患が、ＩｇＧ４関連腎疾患であり、
　前記ラミニンが、ラミニン－５２１である、（３４）に記載のラミニン。
（３７）前記ラミニンが、ラミニンが固定された固相の形態である、（３４）～（３６）のいずれかに記載の使用。
（３８）ＩｇＧ４関連疾患を検査に使用するためのインテグリン。
（３９）前記ＩｇＧ４関連疾患が、自己免疫性膵炎であり、
　前記インテグリンが、インテグリンα６β１である、（３８）に記載のインテグリン。
（４０）前記インテグリンが、インテグリンが固定された固相の形態である、（３８）又は（３９）に記載のインテグリン。
（４０’）自己免疫性膵炎の検査に使用するための、固相に固定された形態の、ラミニン－５１１及びインテグリンα６β１の組み合わせ。
（４１）ＩｇＧ４関連疾患の非ヒトモデル動物を作製する方法に使用するための、ラミニンと免疫学的に反応する抗体。
（４２）ＩｇＧ４関連疾患の非ヒトモデル動物を作製する方法に使用するための、ラミニン。
（４３）ＩｇＧ４関連疾患の非ヒトモデル動物を作製する方法に使用するための、インテグリンと免疫学的に反応する抗体。
（４４）ＩｇＧ４関連疾患の非ヒトモデル動物を作製する方法に使用するための、インテグリン。
（４５）ＩｇＧ４関連疾患の非ヒトモデル動物を作製するための試薬の製造における、ラミニンと免疫学的に反応する抗体の使用。
（４６）ＩｇＧ４関連疾患の非ヒトモデル動物を作製するための試薬の製造における、ラミニンの使用。
（４７）ＩｇＧ４関連疾患の非ヒトモデル動物を作製するための試薬の製造における、インテグリンと免疫学的に反応する抗体の使用。
（４８）ＩｇＧ４関連疾患の非ヒトモデル動物を作製するための試薬の製造における、インテグリンの使用。 The present inventors have surprisingly clarified that the pathogen of IgG4-related disease is an autoantibody against laminin, and that anti-laminin antibody and anti-integrin antibody in serum are indicators of IgG4-related disease. Based on this finding, the inventors have completed the following invention.
(1) A test method for IgG4-related diseases,
Anti-laminin antibody detection step for detecting an antibody that immunologically reacts with laminin as an indicator of IgG4-related disease, and / or immunological reaction with integrin in a sample as an indicator of IgG4-related disease A method comprising an anti-integrin antibody detection step of detecting an antibody to be detected.
(2) the IgG4-related disease is autoimmune pancreatitis,
The method according to (1), wherein the antibody in the anti-laminin antibody detection step is an antibody that immunologically reacts with laminin-511.
(3) the IgG4-related disease is autoimmune pancreatitis,
The method according to (1) or (2), wherein the antibody in the anti-integrin antibody detection step is an antibody that immunologically reacts with integrin α6β1.
(4) The IgG4-related disease is IgG4-related kidney disease,
The method according to (1), wherein the antibody in the anti-laminin antibody detection step is an antibody that immunologically reacts with laminin-521.
(5) The method according to any one of (1) to (4), wherein the anti-laminin antibody detection step comprises detecting the antibody using laminin as an antigen.
(6) The method according to any one of (1) to (5), wherein the anti-integrin antibody detection step includes detecting the antibody using integrin as an antigen.
(7) The method according to any one of (1) to (6), wherein the specimen is a blood sample.
(7 ') A test method for autoimmune pancreatitis,
As an index of autoimmune pancreatitis, an anti-laminin antibody detection step for detecting an antibody immunologically reactive with laminin-511 in a sample blood sample, and as an index of autoimmune pancreatitis in a sample blood sample And an anti-integrin antibody detection step of detecting an antibody that immunologically reacts with integrin α6β1.
(8) A test reagent for testing IgG4-related diseases, including laminin.
(9) The IgG4-related disease is autoimmune pancreatitis,
The test reagent according to (8), wherein the laminin is laminin-511.
(10) The IgG4-related disease is IgG4-related kidney disease,
The test reagent according to (8), wherein the laminin is laminin-521.
(11) The test reagent according to any one of (8) to (10), which contains the laminin in a form fixed to a solid phase.
(12) A test reagent for testing an IgG4-related disease containing integrin.
(13) The IgG4-related disease is autoimmune pancreatitis,
The test reagent according to (12), wherein the integrin is integrin α6β1.
(14) The test reagent according to (12) or (13), comprising the integrin in a form immobilized on a solid phase.
(14 ′) A test reagent for autoimmune pancreatitis comprising laminin-511 and integrin α6β1 in a form immobilized on a solid phase.
(15) A method for evaluating the effect of treatment on an IgG4-related disease,
An anti-laminin antibody detection step of detecting an antibody that immunologically reacts with laminin in a specimen obtained from a test animal treated for an IgG4-related disease; and / or
A method comprising an anti-integrin antibody detection step of detecting an antibody that immunologically reacts with an integrin in a specimen obtained from a test animal treated for an IgG4-related disease.
(16) A method for screening candidate substances for therapeutic agents for IgG4-related diseases,
An anti-laminin antibody detection step for detecting an antibody that immunologically reacts with laminin in a specimen obtained from an animal on which a test substance has been allowed to act;
And a selection step of selecting the test substance as a candidate substance for a therapeutic drug for IgG4-related disease when the antibody in the specimen decreases due to the action of the test substance.
(17) A method for screening candidate substances for therapeutic agents for IgG4-related diseases,
An anti-integrin antibody detection step for detecting an antibody that immunologically reacts with an integrin in a sample obtained from an animal on which a test substance has been allowed to act;
And a selection step of selecting the test substance as a candidate substance for a therapeutic drug for IgG4-related disease when the antibody in the specimen decreases due to the action of the test substance.
(18) A method for producing a non-human model animal of an IgG4-related disease,
A method comprising at least one of an anti-laminin antibody administration step of administering an antibody that immunologically reacts with laminin to a non-human animal, and a laminin immunization step of immunizing a non-human animal with laminin as an antigen.
(19) A method for producing a non-human animal model of an IgG4-related disease,
A method comprising at least one of an anti-integrin antibody administration step of administering an antibody that immunologically reacts with an integrin to a non-human animal, and an integrin immunization step of immunizing the non-human animal with an integrin as an antigen.
(20) A method for obtaining an index of an IgG4-related disease,
A method comprising an anti-laminin antibody detection step of detecting an antibody immunologically reactive with laminin in a sample, and / or an anti-integrin antibody detection step of detecting an antibody immunologically reactive with integrin in the sample.
(21) The IgG4-related disease is autoimmune pancreatitis,
The method according to (20), wherein the antibody in the anti-laminin antibody detection step is an antibody that immunologically reacts with laminin-511.
(22) The IgG4-related disease is autoimmune pancreatitis,
The method according to (20) or (21), wherein the antibody in the anti-integrin antibody detection step is an antibody that immunologically reacts with integrin α6β1.
(23) the IgG4-related disease is IgG4-related kidney disease;
The method according to (20), wherein the antibody in the anti-laminin antibody detection step is an antibody that immunologically reacts with laminin-521.
(24) The method according to any one of (20) to (23), wherein the anti-laminin antibody detection step comprises detecting the antibody using laminin as an antigen.
(25) The method according to any one of (20) to (24), wherein the anti-integrin antibody detection step comprises detecting the antibody using integrin as an antigen.
(26) The method according to any one of (20) to (25), wherein the specimen is a blood sample separated from a test animal.
(26 ′) A method for obtaining an index of autoimmune pancreatitis,
A method comprising an anti-laminin antibody detection step for detecting an antibody immunologically reactive with laminin-511 in a sample, and an anti-integrin antibody detection step for detecting an antibody immunologically reactive with integrin α6β1 in the sample .
(27) Use of laminin in the manufacture of a test reagent for testing an IgG4-related disease.
(28) The IgG4-related disease is autoimmune pancreatitis,
The use according to (27), wherein the laminin is laminin-511.
(29) The IgG4-related disease is IgG4-related kidney disease,
The use according to (27), wherein the laminin is laminin-521.
(30) The use according to any one of (27) to (29), wherein the laminin is in the form of a solid phase on which laminin is immobilized.
(31) Use of an integrin in the manufacture of a test reagent for testing an IgG4-related disease.
(32) The IgG4-related disease is autoimmune pancreatitis,
The use according to (31), wherein the integrin is integrin α6β1.
(33) The use according to (31) or (32), wherein the integrin is in the form of a solid phase on which the integrin is immobilized.
(33 ′) Use of laminin-511 and integrin α6β1 in a form immobilized on a solid phase in the manufacture of a test reagent for autoimmune pancreatitis.
(34) Laminin for use in testing for IgG4-related diseases.
(35) The IgG4-related disease is autoimmune pancreatitis,
The laminin according to (34), wherein the laminin is laminin-511.
(36) The IgG4-related disease is IgG4-related kidney disease,
The laminin according to (34), wherein the laminin is laminin-521.
(37) The use according to any one of (34) to (36), wherein the laminin is in the form of a solid phase on which laminin is immobilized.
(38) An integrin for use in testing IgG4-related diseases.
(39) The IgG4-related disease is autoimmune pancreatitis,
The integrin according to (38), wherein the integrin is integrin α6β1.
(40) The integrin according to (38) or (39), wherein the integrin is in the form of a solid phase on which the integrin is immobilized.
(40 ′) A combination of laminin-511 and integrin α6β1 in a form immobilized on a solid phase for use in testing for autoimmune pancreatitis.
(41) An antibody that immunologically reacts with laminin for use in a method for producing a non-human model animal of an IgG4-related disease.
(42) Laminin for use in a method for producing a non-human model animal of an IgG4-related disease.
(43) An antibody that immunologically reacts with an integrin for use in a method for producing a non-human model animal of an IgG4-related disease.
(44) An integrin for use in a method for producing a non-human model animal of an IgG4-related disease.
(45) Use of an antibody that immunologically reacts with laminin in the manufacture of a reagent for producing a non-human model animal of an IgG4-related disease.
(46) Use of laminin in the manufacture of a reagent for producing a non-human model animal of an IgG4-related disease.
(47) Use of an antibody that immunologically reacts with integrin in the manufacture of a reagent for producing a non-human model animal of an IgG4-related disease.
(48) Use of an integrin in the manufacture of a reagent for producing a non-human model animal of an IgG4-related disease.
　本明細書は本願の優先権の基礎となる日本国特許出願番号２０１６－１４２７０１号の開示内容を包含する。 This specification includes the disclosure of Japanese Patent Application No. 2016-142701 which is the basis of the priority of the present application.
　本発明の１つ以上の実施形態によれば、ＩｇＧ４関連疾患の検査方法、ＩｇＧ４関連疾患を検査するための検査試薬、ＩｇＧ４関連疾患に対する治療の効果を評価する方法、ＩｇＧ４関連疾患の治療薬の候補物質をスクリーニングする方法として有効な手段が提供される。 According to one or more embodiments of the present invention, a method for testing an IgG4-related disease, a test reagent for testing an IgG4-related disease, a method for evaluating the effect of a treatment on an IgG4-related disease, a therapeutic agent for an IgG4-related disease, An effective means is provided as a method for screening candidate substances.
図１Ａは、実験１での、対照ＩｇＧ投与マウス（左列）と、患者ＩｇＧ投与マウス（右列）の、ＩｇＧ投与から１２時間後の膵臓のＨ＆Ｅ染色（上段）と、Ｇｒ１の染色（下段）の結果を示す。観察像中のスケールバーは上段では５０μｍ、下段では２０μｍを示す。FIG. 1A shows H & E staining of pancreas 12 hours after IgG administration (upper row) and Gr1 staining (lower row) of control IgG-treated mice (left column) and patient IgG-treated mice (right column) in Experiment 1. ) Result. The scale bar in the observed image shows 50 μm in the upper stage and 20 μm in the lower stage. 図１Ｂは、実験１での、対照ＩｇＧ（三角）及び患者ＩｇＧ（丸）の投与量と、浮腫面積との相関関係を示す。FIG. 1B shows the correlation between the dose of control IgG (triangle) and patient IgG (circle) and the edema area in Experiment 1. 図１Ｃは、実験１での、対照ＩｇＧ投与マウス（左）と、患者ＩｇＧ投与マウス（右）の、ＩｇＧ投与から１２時間後の膵臓の壊死又は出血の病理組織的グレードと、Ｇｒ１陽性細胞数を示す（＊ｐ＜０．０５、＊＊ｐ＜０．００５：ｐａｉｒｅｄ　Ｓｔｕｄｅｎｔ’ｓ　ｔｅｓｔによる）。FIG. 1C shows the histopathological grade of necrosis or hemorrhage of pancreas 12 hours after IgG administration and the number of Gr1 positive cells in control IgG-treated mice (left) and patient IgG-treated mice (right) in Experiment 1. ( * P <0.05, ** p <0.005: according to paired Student's test). 図２Ａは、実験１での、対照ＩｇＧ投与マウス（左列）と、患者ＩｇＧ投与マウス（右列）の、ＩｇＧ投与から１２時間後の膵臓でのＩｇＧ（上段）、ＩｇＧ１（中段）、ＩｇＧ４（下段）の免疫組織化学的染色の結果を示す。観察像中のスケールバーは２０μｍを示す。FIG. 2A shows IgG in the pancreas 12 hours after IgG administration (upper row), IgG1 (middle row), IgG4 of control IgG-treated mice (left column) and patient IgG-treated mice (right column) in Experiment 1. The result of immunohistochemical staining of (lower) is shown. The scale bar in the observed image indicates 20 μm. 図２Ｂは、実験１での、対照ＩｇＧ投与マウスと患者ＩｇＧ投与マウスの、膵臓組織の免疫蛍光染色の結果を示す。観察像中のスケールバーは２０μｍを示す。FIG. 2B shows the results of immunofluorescence staining of pancreatic tissue in control IgG-treated mice and patient IgG-treated mice in Experiment 1. The scale bar in the observed image indicates 20 μm. 図３Ａの上段の「対照１」は、１０名の対照のうち特定の１名（対照１）から得た対照ＩｇＧ１及び対照ＩｇＧ４の一方又は両方を投与したマウスの投与１２時間後の膵臓のＨ＆Ｅ染色の観察像である。観察像中のスケールバーは２０μｍを示す。図３Ａの中段及び下段「患者９」は、１０名のＩｇＧ４関連疾患患者のうち特定の１名（患者９）から得た患者ＩｇＧ１及び患者ＩｇＧ４の一方又は両方を投与したマウスの投与１２時間後の膵臓の、Ｈ＆Ｅ染色の観察像（中段）、及び、Ｇｒ１の免疫組織化学的染色の観察像（下段）である。観察像中のスケールバーは２０μｍを示す。“Control 1” in the upper part of FIG. 3A shows pancreatic H & E 12 hours after administration of mice administered with one or both of control IgG1 and control IgG4 obtained from a specific one of the 10 controls (control 1). It is an observation image of dyeing | staining. The scale bar in the observed image indicates 20 μm. The middle and lower “patient 9” in FIG. 3A shows 12 hours after administration of a mouse administered with one or both of patient IgG1 and patient IgG4 from one specific patient (patient 9) among 10 IgG4-related disease patients. 2 is an observation image of H & E staining (middle stage) and an observation image of immunohistochemical staining of Gr1 (lower stage). The scale bar in the observed image indicates 20 μm. 図３Ｂは、対照ＩｇＧ１及び対照ＩｇＧ４の一方又は両方を投与したマウス（左）と、患者ＩｇＧ１及び患者ＩｇＧ４の一方又は両方を投与したマウス（右）の、投与から１２時間後の膵臓の壊死又は出血の病理組織的グレードと、Ｇｒ１陽性細胞数を示す（＊ｐ＜０．０５：ｐａｉｒｅｄ　Ｓｔｕｄｅｎｔ’ｓ　ｔｅｓｔによる）。FIG. 3B shows pancreatic necrosis or 12 hours after administration of mice that received one or both of control IgG1 and control IgG4 (left) and mice that received one or both of patient IgG1 and patient IgG4 (right). The histopathological grade of bleeding and the number of Gr1 positive cells are shown ( * p <0.05: according to Paired Student's test). 図３Ｃは、１０名のＩｇＧ４関連疾患患者のうち特定の１名（患者１）から得た患者ＩｇＧ１及び患者ＩｇＧ４の一方又は両方を投与したマウスの投与１２時間後の膵臓の、ヒトＩｇＧ１又はヒトＩｇＧ４の免疫組織化学的染色の観察像である。観察像中のスケールバーは２０μｍを示す。FIG. 3C shows pancreatic human IgG1 or human 12 hours after administration of mice administered with one or both of patient IgG1 and patient IgG4 from one particular patient (patient 1) out of 10 IgG4-related diseases It is an observation image of immunohistochemical staining of IgG4. The scale bar in the observed image indicates 20 μm. 図４Ａ上段左は、ＡＩＰ病変のある部位（左側）と、病変のない部位（右側）との境界部分から採取した、ＡＩＰ患者の膵臓組織切片をＨ＆Ｅ染色した観察像を示す。図４Ａ上段右は、同様の膵臓組織切片の、ＩｇＧ４に対する免疫組織化学的染色の観察像を示す。スケールバーは２００μｍを示す。図４Ａ下段左は、図４Ａ上段右の観察像のうち、病変のある腺房の部分を拡大した観察像である。図４Ａ下段中は、図４Ａ上段右の観察像のうち、病変のある小葉間隙の部分を拡大した観察像である。図４Ａ下段右は、図４Ａ上段右の観察像のうち、病変のない正常な腺房の部分を拡大した観察像である。図４Ａ下段の各観察像においてスケールバーは２０μｍを示す。The upper left of FIG. 4A shows an observation image obtained by H & E staining of a pancreatic tissue section of an AIP patient collected from a boundary portion between a site with AIP lesion (left side) and a site without lesion (right side). The upper right of FIG. 4A shows an observation image of immunohistochemical staining for IgG4 of the same pancreatic tissue section. The scale bar indicates 200 μm. The lower left of FIG. 4A is an enlarged image of the lesioned acinar portion in the upper right image of FIG. 4A. The lower part of FIG. 4A is an observation image obtained by enlarging the lesioned leaflet gap portion in the upper right part of FIG. 4A. The lower right side of FIG. 4A is an observation image obtained by enlarging a normal acinar portion without a lesion in the upper right image of FIG. 4A. In each observed image in the lower part of FIG. 4A, the scale bar indicates 20 μm. 図４Ｂの各像は、ＡＩＰ患者の膵臓での病変のある腺房を免疫蛍光染色した観察像を示す。図４Ｂの各像ではスケールバーは２０μｍを示す。図４Ｂ上段左は、ＡＩＰ患者の膵臓での病変のある腺房の、免疫蛍光染色されたＩｇＧ４の観察像を示す。図４Ｂ上段中は、図４Ｂ上段左と同じ観察領域の、免疫蛍光染色されたアミラーゼの観察像を示す。図４Ｂ上段右は、図４Ｂ上段左のＩｇＧ４の観察像と、図４Ｂ上段中のアミラーゼの観察像とを重ね合わせた像である。図４Ｂ下段左は、ＡＩＰ患者の膵臓での病変のある腺房の、免疫蛍光染色されたＩｇＧ４の観察像を示す。図４Ｂ下段中は、図４Ｂ下段左と同じ観察領域の、免疫蛍光染色されたコラーゲンＩＶの観察像を示す。図４Ｂ下段右は、図４Ｂ下段左のＩｇＧ４の観察像と、図４Ｂ下段中のコラーゲンＩＶの観察像とを重ね合わせた像である。Each image in FIG. 4B shows an observation image obtained by immunofluorescent staining of a lesioned acinus in the pancreas of an AIP patient. In each image of FIG. 4B, the scale bar indicates 20 μm. The upper left of FIG. 4B shows an observed image of IgG4 stained with immunofluorescence in an acinus with lesions in the pancreas of an AIP patient. The upper part of FIG. 4B shows an observation image of amylase stained with immunofluorescence in the same observation area as the upper left part of FIG. 4B. The upper right of FIG. 4B is an image obtained by superimposing the observed image of IgG4 on the upper left of FIG. 4B and the observed image of amylase in the upper part of FIG. 4B. The lower left of FIG. 4B shows an observed image of IgG4 stained with immunofluorescence in an acinus with lesions in the pancreas of an AIP patient. The lower part of FIG. 4B shows an observation image of collagen IV stained with immunofluorescence in the same observation area as the lower left part of FIG. 4B. The lower right of FIG. 4B is an image obtained by superimposing the observed image of IgG4 on the lower left of FIG. 4B and the observed image of collagen IV in the lower part of FIG. 4B. 図４Ｃの各像は、ＡＩＰ患者の膵臓での病変のある小葉間隙を免疫蛍光染色した観察像を示す。図４Ｃの各像ではスケールバーは２０μｍを示す。図４Ｃ左は、ＡＩＰ患者の膵臓での病変のある小葉間隙の、免疫蛍光染色されたＩｇＧ４の観察像を示す。図４Ｃ中は、図４Ｃ左と同じ観察領域の、免疫蛍光染色されたコラーゲンＩＶの観察像を示す。図４Ｃ右は、図４Ｃ左のＩｇＧ４の観察像と、図４Ｃ中のコラーゲンＩＶの観察像とを重ね合わせた像である。Each image in FIG. 4C shows an observation image obtained by immunofluorescent staining of a lobular space with a lesion in the pancreas of an AIP patient. In each image of FIG. 4C, the scale bar indicates 20 μm. The left side of FIG. 4C shows an observed image of immunofluorescent-stained IgG4 in a lobular space with lesions in the pancreas of an AIP patient. FIG. 4C shows an observation image of collagen IV stained with immunofluorescence in the same observation region as the left of FIG. 4C. The right side of FIG. 4C is an image obtained by superimposing the observed image of IgG4 on the left side of FIG. 4C and the observed image of collagen IV in FIG. 4C. ＡＩＰ患者血清及び対照血清中の抗ヒトラミニン－５１１活性断片抗体の濃度を示す。The concentration of anti-human laminin-511 active fragment antibody in AIP patient serum and control serum is shown. ＡＩＰ患者血清及び対照血清中の抗ヒトコラーゲンＩＶ抗体の濃度を示す。The concentration of anti-human collagen IV antibody in AIP patient serum and control serum is shown. ＡＩＰ患者血清及び対照血清中の抗ヒトフィブロネクチン抗体の濃度を示す。The concentration of anti-human fibronectin antibody in AIP patient serum and control serum is shown. ＡＩＰ患者血清及び対照血清中の抗ＧＦＰ抗体の濃度を示す。The concentration of anti-GFP antibody in AIP patient serum and control serum is shown. 図６は、Ｈ＆Ｅ染色により染色した膵臓の組織切片の観察像を示す。図６左が対照群の膵臓の観察像、図６右がラミニン－５１１により免疫した試験群の膵臓の観察像である。FIG. 6 shows an observation image of a tissue section of the pancreas stained by H & E staining. FIG. 6 left is an observation image of the pancreas of the control group, and FIG. 6 right is an observation image of the pancreas of the test group immunized with laminin-511. ＡＩＰ患者血清及び対照血清中の抗ヒトラミニン－５２１抗体の濃度を示す。The concentration of anti-human laminin-521 antibody in AIP patient serum and control serum is shown. 図８左側にＩｇＧ４関連腎疾患患者ＩｇＧを皮下投与したマウスの腎臓の観察像を示し、図８右側に対照ＩｇＧを皮下投与したマウスの腎臓のヒトＩｇＧ４染色観察像を示す。上段は、腎臓全体のヒトＩｇＧ４染色観察像を示し、下段は糸状体のヒトＩｇＧ４染色観察像を示す。The left side of FIG. 8 shows an observation image of the kidney of a mouse subcutaneously administered with IgG4 related kidney disease patient IgG, and the right side of FIG. 8 shows an observation image of human IgG4 staining of the kidney of a mouse subcutaneously administered with control IgG. The upper row shows the human IgG4 staining observation image of the whole kidney, and the lower row shows the human IgG4 staining observation image of the filamentous body. 実験８における、トレーニング群である１０名のＡＩＰ患者及び１０名の対照からの血清試料の、抗ヒトラミニン－５１１－Ｅ８　ＩｇＧ抗体レベルの測定結果を示す。The measurement results of anti-human laminin-511-E8 IgG antibody levels of serum samples from 10 AIP patients and 10 controls in the training group in Experiment 8 are shown. 実験８における、バリデーション群である４１名のＡＩＰ患者及び１０２名の対照（健常者２０名、ＡＩＰ以外の疾患患者８２名）からの血清試料の、抗ヒトラミニン－５１１－Ｅ８　ＩｇＧ抗体レベルの測定結果を示す。Results of measurement of anti-human laminin-511-E8 IgG antibody levels in serum samples from 41 AIP patients and 102 controls (20 healthy subjects, 82 non-AIP disease patients) in the validation group in Experiment 8 Indicates. 実験９における、２６名の抗ラミニン５１１－Ｅ８抗体陽性ＡＩＰ患者、２５名の抗ラミニン５１１－Ｅ８抗体陰性ＡＩＰ患者、及び、１１２名の対照からの血清試料での、抗インテグリンα６β１　ＩｇＧ抗体レベルの測定結果を示す。Anti-integrin α6β1 IgG antibody levels in serum samples from 26 anti-laminin 511-E8 antibody positive AIP patients, 25 anti-laminin 511-E8 antibody negative AIP patients, and 112 controls in Experiment 9. The measurement results are shown. 実験９における、２６名の抗ラミニン５１１－Ｅ８抗体陽性ＡＩＰ患者、２５名の抗ラミニン５１１－Ｅ８抗体陰性ＡＩＰ患者、及び、１１２名の対照からの血清試料での、抗インテグリンα３β１　ＩｇＧ抗体レベルの測定結果を示す。Anti-integrin α3β1 IgG antibody levels in serum samples from 26 anti-laminin 511-E8 antibody positive AIP patients, 25 anti-laminin 511-E8 antibody negative AIP patients, and 112 controls in Experiment 9. The measurement results are shown. 図１２上段は、抗ラミニン５１１－Ｅ８抗体陽性ＡＩＰ患者の造影ＣＴによる膵画像である。図１２下段は、抗インテグリンα６β１抗体陽性ＡＩＰ患者の造影ＣＴによる膵画像である。各画像中の矢印は病変箇所を指す。The upper part of FIG. 12 is a pancreatic image obtained by contrast CT of an anti-laminin 511-E8 antibody positive AIP patient. The lower part of FIG. 12 is a pancreatic image obtained by contrast CT of an anti-integrin α6β1 antibody-positive AIP patient. The arrow in each image indicates the lesion site. 抗ラミニン５１１－Ｅ８抗体陽性ＡＩＰ患者５名の、ステロイド（プレドニゾン）を用いた治療の前後での、血清中の抗ラミニン５１１－Ｅ８抗体レベルの測定結果を示す。The measurement results of anti-laminin 511-E8 antibody levels in serum before and after treatment with steroid (prednisone) of 5 patients with anti-laminin 511-E8 antibody positive AIP are shown.
＜１．ＩｇＧ４関連疾患＞
　本発明においてＩｇＧ４関連疾患とは、血清ＩｇＧ４高値とＩｇＧ４陽性形質細胞の組織浸潤または腫瘤形成を特徴とする疾患群であり、具体的には、自己免疫性膵炎（ＡＩＰ）、ＩｇＧ４関連腎疾患、ＩｇＧ４関連涙腺唾液腺炎、ＩｇＧ４関連硬化性胆管炎、ＩｇＧ４関連眼疾患、ＩｇＧ４関連動脈周囲炎、ＩｇＧ４関連下垂体炎、ＩｇＧ４関連肺疾患、ＩｇＧ４関連後腹膜線維症が例示できる。
＜２．ＩｇＧ４関連疾患の検査方法＞
　本発明は第一に、
　ＩｇＧ４関連疾患の検査方法であって、
　ＩｇＧ４関連疾患の指標として、検体中の、ラミニンと免疫学的に反応する抗体を検出する抗ラミニン抗体検出工程、及び／又は
　ＩｇＧ４関連疾患の指標として、検体中の、インテグリンと免疫学的に反応する抗体を検出する抗インテグリン抗体検出工程
を含む方法に関する。 <1. IgG4-related disease>
In the present invention, the IgG4-related disease is a disease group characterized by tissue infiltration or tumor formation of high serum IgG4 and IgG4-positive plasma cells, specifically, autoimmune pancreatitis (AIP), IgG4-related kidney disease, Examples include IgG4-related lacrimal gland salivary gland, IgG4-related sclerosing cholangitis, IgG4-related eye disease, IgG4-related periarteritis, IgG4-related hypophysitis, IgG4-related lung disease, IgG4-related retroperitoneal fibrosis.
<2. Method for testing IgG4-related disease>
The present invention firstly
A test method for IgG4-related diseases,
Anti-laminin antibody detection step for detecting an antibody that immunologically reacts with laminin as an indicator of IgG4-related disease, and / or immunological reaction with integrin in a sample as an indicator of IgG4-related disease The present invention relates to a method comprising an anti-integrin antibody detection step of detecting an antibody to be detected.
　本発明の検査方法の１つ以上の実施形態は、ラミニンに対する抗体（自己抗体）が、ＩｇＧ４関連疾患の病原物質であり、被検動物から取得した検体中の前記抗体が、ＩｇＧ４関連疾患に罹患していることの指標となるという驚くべき知見に基づき完成された。 In one or more embodiments of the test method of the present invention, the antibody against laminin (autoantibody) is a pathogen of an IgG4-related disease, and the antibody in a specimen obtained from a test animal suffers from an IgG4-related disease. It was completed based on the surprising finding that it is an indicator of what is being done.
　また、本発明の検査方法の１つ以上の実施形態は、ラミニンとインテグリンとの結合が、ＩｇＧ４関連疾患の病原物質のターゲットであり、被検動物から取得した検体中の抗ラミニン抗体及び／又は抗インテグリン抗体が、ＩｇＧ４関連疾患に罹患していることの指標となるという驚くべき知見に基づき完成された。 In one or more embodiments of the test method of the present invention, the binding between laminin and integrin is a target of a pathogen of an IgG4-related disease, and an anti-laminin antibody in a specimen obtained from a test animal and / or It was completed based on the surprising finding that anti-integrin antibodies are indicative of having an IgG4-related disease.
　本発明の検査方法が対象とする被検動物は特に限定されずヒトであってもよいし、他の非ヒト哺乳動物であってもよいが、好ましくはヒトである。 The test animal targeted by the test method of the present invention is not particularly limited and may be a human or other non-human mammal, but is preferably a human.
　本発明の検査方法に用いる検体としては、被検動物から採取される体液が挙げられる。具体的には、血清、血漿、全血等の血液試料のほか、唾液、髄液、尿などの血液以外の体液試料であってもよい。また、体液に限らず、被検動物から採取される組織、例えば、検査しようとするＩｇＧ４関連疾患の疾患部位（例えば膵臓、腎臓、唾液腺等）から採取される組織も、検体として用いることができる。検体は、被検動物から分離された形態で本発明の検査方法に用いる。 Specimens used in the test method of the present invention include body fluids collected from test animals. Specifically, in addition to blood samples such as serum, plasma and whole blood, body fluid samples other than blood such as saliva, spinal fluid and urine may be used. Further, not only body fluids but also tissues collected from test animals, for example, tissues collected from disease sites of IgG4-related diseases to be examined (for example, pancreas, kidney, salivary gland, etc.) can be used as specimens. . The specimen is used in the examination method of the present invention in a form separated from the subject animal.
　ラミニンは、天然型では、α鎖、β鎖およびγ鎖の３本のサブユニット鎖からなる巨大なヘテロ３量体分子である。α鎖としてα１～α５、β鎖としてβ１～β３、γ鎖としてγ１～γ３が知られており、それらの組み合わせで１２種類以上のアイソフォームが存在する（表１参照）。本発明においてラミニンのアイソフォームは限定されない。すなわち検出しようとする抗体が免疫学的に反応するラミニンは、α１～α５から選択される１種のα鎖、β１～β３から選択される１種のβ鎖、γ１～γ３から選択される１種のγ鎖からなるものであることができ、具体的には、表１に記載の１２種類のいずれか、或いは他のアイソフォームであることができ、好ましくはα鎖がα５、β鎖がβ１、γ鎖がγ１である「ラミニン－５１１」、及び、α鎖がα５、β鎖がβ２、γ鎖がγ１である「ラミニン－５２１」である。 Laminin is a huge heterotrimeric molecule consisting of three subunit chains of α chain, β chain and γ chain in the natural type. α1 to α5 are known as α chains, β1 to β3 are known as β chains, and γ1 to γ3 are known as γ chains, and combinations thereof include 12 or more isoforms (see Table 1). In the present invention, laminin isoforms are not limited. That is, the laminin to which the antibody to be detected immunologically reacts is selected from one α chain selected from α1 to α5, one β chain selected from β1 to β3, and γ1 to γ3 1 It can consist of γ chains of species, specifically, can be any of the 12 types listed in Table 1 or other isoforms, preferably α chain is α5, β chain is “Laminin-511” in which β1 and γ chain are γ1, and “Laminin-521” in which α chain is α5, β chain is β2, and γ chain is γ1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　本発明において、検出しようとする抗体が免疫学的に反応するラミニンの起源は特に限定されないが、ＩｇＧ４関連疾患は通常は自己免疫疾患であることから、被検動物と同じ種のラミニンであることが好ましく、特にヒトが好ましい。ヒト等の哺乳動物の種のラミニンのα鎖、β鎖、γ鎖をコードする遺伝子の塩基配列情報および各鎖のアミノ酸配列情報は、公知のデータベース（ＧｅｎＢａｎｋ等）から取得することができる。表２に、ヒトを含む主な哺乳類について、ラミニンを構成する各鎖のアクセッション番号を示す。表２に示すアミノ酸配列を有するラミニンの各鎖が、更に翻訳後修飾を受けたものが、ラミニンを形成してもよい。これら以外の各種哺乳動物のラミニン構成鎖の塩基配列情報およびアミノ酸配列情報も同様に公知のデータベース（ＧｅｎＢａｎｋ等）から取得することができる。 In the present invention, the origin of laminin to which the antibody to be detected immunologically reacts is not particularly limited. However, since an IgG4-related disease is usually an autoimmune disease, it must be the same kind of laminin as the test animal. Are preferred, and humans are particularly preferred. The nucleotide sequence information of the genes encoding the α chain, β chain, and γ chain of laminin from mammalian species such as humans and the amino acid sequence information of each chain can be obtained from a known database (GenBank, etc.). Table 2 shows the accession numbers of the chains constituting laminin for main mammals including humans. The laminin having the amino acid sequence shown in Table 2 may be further subjected to post-translational modification to form laminin. Other than these, the base sequence information and amino acid sequence information of laminin constituent chains of various mammals can also be obtained from known databases (GenBank, etc.).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　本発明においてラミニン、ラミニン－５１１、ラミニン－５２１という各用語は、特に限定しない場合は、α鎖、β鎖及びγ鎖がそれぞれ全長からなる天然型の形態には限定されず、それぞれ、ラミニンの断片、ラミニン－５１１の断片、ラミニン－５２１の断片や、天然型のラミニン、天然型のラミニン－５１１、天然型のラミニン－５２１と等価な形態のものを包含する。従って、検出しようとする抗体は、ラミニンの断片と免疫学的に反応するものであってもよい。ラミニンの断片としては、天然型のラミニンの３量体を構成するα鎖、β鎖、γ鎖のうち少なくとも１つが全長よりも短いものが挙げられる。ラミニンの断片は、３量体を形成していることが好ましく、インテグリン結合活性を有していることがより好ましい。ラミニンの断片が３量体を形成していることは、ラミニンの断片をＳＤＳ－ＰＡＧＥに供し、バンドの数を検出すること等により確認できる。ラミニンの断片がインテグリン結合活性を有していることは、ＥＬＩＳＡ法等により確認することができる。 In the present invention, the terms laminin, laminin-511, and laminin-521 are not particularly limited to natural forms in which the α chain, the β chain, and the γ chain are all full lengths, respectively. Fragments, fragments of laminin-511, fragments of laminin-521, natural laminin, natural laminin-511, and forms equivalent to natural laminin-521 are included. Therefore, the antibody to be detected may be one that immunologically reacts with a laminin fragment. Examples of the laminin fragment include those in which at least one of the α chain, β chain, and γ chain constituting the natural laminin trimer is shorter than the full length. The laminin fragment preferably forms a trimer, and more preferably has integrin binding activity. It can be confirmed that the laminin fragment forms a trimer by subjecting the laminin fragment to SDS-PAGE and detecting the number of bands. It can be confirmed by ELISA or the like that the laminin fragment has integrin binding activity.
　３量体を形成し、且つ、インテグリン結合活性を有するラミニンの断片としては、天然型のラミニンをエラスターゼ等のタンパク質分解酵素で消化して得られる断片が挙げられ、特に好ましくはＥ８フラグメントである。ラミニンのＥ８フラグメントは、α鎖のＣ末端断片から、Ｃ末端の球状（Ｇ）ドメイン４および５が除かれた断片（α鎖Ｅ８）、β鎖のＣ末端断片（β鎖Ｅ８）およびγ鎖のＣ末端断片（γ鎖Ｅ８）が３量体を形成したフラグメントである（Hiroyuki Ido, Aya Nakamura, Reiko Kobayashi, Shunsuke Ito, Shaoliang Li, Sugiko Futaki, and Kiyotoshi Sekiguchi, The Journal of Biological Chemistry, 282, 11144-11154, 2007）。ラミニンのＥ８フラグメントは、全長ラミニンを膵エラスターゼにより酵素的に消化して得ることができる。市販のラミニンのＥ８フラグメントとしては、ヒトラミニン－５１１－Ｅ８（８９２０１２，株式会社ニッピ製）が例示できる。 Examples of the fragment of laminin that forms a trimer and has integrin binding activity include a fragment obtained by digesting natural laminin with a proteolytic enzyme such as elastase, and particularly preferably an E8 fragment. The E8 fragment of laminin is obtained by removing the C-terminal globular (G) domains 4 and 5 from the α-chain C-terminal fragment (α-chain E8), β-chain C-terminal fragment (β-chain E8) and γ-chain. The C-terminal fragment (γ-chain E8) is a fragment that forms a trimer (Hiroyuki Ido, Aya Nakamura, Reiko Kobayashi, Shunsuke Ito, Shaoliang Li, Sugiko Futaki, and Kiyotoshi Sekiguchi, The Journal of Biological Chemist 11144-11154, 2007). The E8 fragment of laminin can be obtained by enzymatic digestion of full length laminin with pancreatic elastase. An example of a commercially available E8 fragment of laminin is human laminin-511-E8 (892012, manufactured by Nippi Co., Ltd.).
　インテグリンは、天然型では、α鎖及びβ鎖の２本のサブユニット鎖からなるヘテロ２量体分子である。α鎖としてα１～α１１、αＶ、αＸ、αＭ、αＬ、αＤ、αＥ、αＩＩｂ、β鎖としてβ１～β８が知られており、それらの組み合わせが異なる複数のアイソフォームが存在する。ラミニンのリガンドとなり得るインテグリンのアイソフォームとしては、インテグリンα６β１、インテグリンα３β１、インテグリンα６β４が知られている。本発明においてインテグリンのアイソフォームは限定されないが、好ましくはα鎖がα６、β鎖がβ１である「インテグリンα６β１」である。 Integrin is a heterodimer molecule consisting of two subunit chains of α chain and β chain in the natural type. α1 to α11, αV, αX, αM, αL, αD, αE, αIIb as β chains, β1 to β8 as β chains, and a plurality of isoforms having different combinations thereof exist. Integrin α6β1, integrin α3β1, and integrin α6β4 are known as integrin isoforms that can be laminin ligands. In the present invention, the integrin isoform is not limited, but is preferably “integrin α6β1” in which the α chain is α6 and the β chain is β1.
　本発明において、検出しようとする抗体が免疫学的に反応するインテグリンの起源は特に限定されないが、ＩｇＧ４関連疾患は通常は自己免疫疾患であることから、被検動物と同じ種のインテグリンであることが好ましく、特にヒトが好ましい。ヒト等の哺乳動物の種のインテグリンのα鎖、β鎖をコードする遺伝子の塩基配列情報および各鎖のアミノ酸配列情報は、公知のデータベース（ＧｅｎＢａｎｋ等）から取得することができる。表３に、ヒトインテグリンを構成する各鎖のアミノ酸配列及び塩基配列のアクセッション番号を示す。ヒトインテグリンのα６鎖及びβ１鎖には、それぞれ、３つのスプライシングバリアントが知られている。表３に示すアミノ酸配列を有するヒトインテグリンのα鎖、β鎖が、更に翻訳後修飾を受けたものが、ヒトインテグリンを形成してもよい。これら以外の各種哺乳動物のインテグリン構成鎖の塩基配列情報およびアミノ酸配列情報も同様に公知のデータベース（ＧｅｎＢａｎｋ等）から取得することができる。 In the present invention, the origin of the integrin to which the antibody to be detected reacts immunologically is not particularly limited. However, since an IgG4-related disease is usually an autoimmune disease, it is an integrin of the same species as the test animal. Are preferred, and humans are particularly preferred. The nucleotide sequence information of the genes encoding integrin α chain and β chain of mammalian species such as humans and the amino acid sequence information of each chain can be obtained from a known database (GenBank, etc.). Table 3 shows the amino acid sequence and base sequence accession number of each chain constituting human integrin. Three splicing variants are known for each of α6 chain and β1 chain of human integrin. Human integrin having the amino acid sequence shown in Table 3 that has undergone post-translational modification may also form human integrin. The base sequence information and amino acid sequence information of integrin constituent chains of various mammals other than these can also be obtained from known databases (GenBank etc.).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　本発明においてインテグリン、インテグリンα６β１、インテグリンα３β１という各用語は、特に限定しない場合は、α鎖及びβ鎖がそれぞれ全長からなる形態には限定されず、それぞれ、インテグリンの断片、インテグリンα６β１の断片、インテグリンα３β１の断片や、天然型のインテグリン、天然型のインテグリンα６β１、天然型のインテグリンα３β１と等価な形態のものを包含する。従って、検出しようとする抗体は、インテグリンの断片と免疫学的に反応するものであってもよい。インテグリンの断片としては、インテグリンの２量体を構成するα鎖、β鎖のうち少なくとも１つが全長よりも短いものが挙げられる。インテグリンの断片は、２量体を形成していることが好ましく、ラミニン結合活性を有していることがより好ましい。インテグリンの断片が２量体を形成していることは、インテグリンの断片をＳＤＳ－ＰＡＧＥに供し、バンドの数を検出すること等により確認できる。インテグリンの断片がラミニン結合活性を有していることは、ＥＬＩＳＡ法等により確認することができる。市販のインテグリンとしては、リコンビナントヒトインテグリンα６β１（Ｒ＆ＤＳｙｓｔｅｍｓ、米国ミネソタ州、製品番号７８０９－Ａ６）、リコンビナントヒトインテグリンα３β１（Ｒ＆ＤＳｙｓｔｅｍｓ、米国ミネソタ州、製品番号２８４０－Ａ３）が例示できる。 In the present invention, the terms integrin, integrin α6β1, and integrin α3β1 are not particularly limited to forms in which the α chain and β chain are each composed of the full length, respectively, and are each an integrin fragment, integrin α6β1 fragment, integrin It includes fragments of α3β1, natural integrin, natural integrin α6β1, and forms equivalent to natural integrin α3β1. Therefore, the antibody to be detected may be one that immunologically reacts with the integrin fragment. Examples of integrin fragments include those in which at least one of the α chain and β chain constituting the integrin dimer is shorter than the full length. The integrin fragment preferably forms a dimer, and more preferably has laminin binding activity. The fact that the integrin fragment forms a dimer can be confirmed by subjecting the integrin fragment to SDS-PAGE and detecting the number of bands. It can be confirmed by ELISA or the like that the integrin fragment has laminin binding activity. Examples of commercially available integrins include recombinant human integrin α6β1 (R & D Systems, Minnesota, USA, product number 7809-A6) and recombinant human integrin α3β1 (R & D Systems, Minnesota, USA, product number 2840-A3).
　インテグリンの断片としては、市販されているヒトインテグリンα６β１の断片であるＨｕｍａｎ　ＩＴＧＡ６　＆　ＩＴＧＢ１（中国北京、Ｓｉｎｏ　Ｂｉｏｌｏｇｉｃａｌ、製品番号ＣＴ０１３－Ｈ２５０８Ｈ－２０）が例示できる。 Examples of integrin fragments include Human ITGA6 & ITGB1 (Beijing China, Sino Biological, product number CT013-H2508H-20), which is a commercially available fragment of human integrin α6β1.
　抗ラミニン抗体検出工程は、検体中の、ラミニンと免疫学的に反応する抗体を検出する工程である。抗インテグリン抗体検出工程は、検体中の、インテグリンと免疫学的に反応する抗体を検出する工程である。各工程において「検出」とは、検体中に、ラミニン又はインテグリンと免疫学的に反応する抗体が存在するか否かを確認することのほか、検体中における該抗体の含有量を測定すること、すなわち「定量」すること、も包含する概念である。 The anti-laminin antibody detection step is a step of detecting an antibody that immunologically reacts with laminin in a sample. The anti-integrin antibody detection step is a step of detecting an antibody that immunologically reacts with the integrin in the specimen. In each step, “detection” refers to whether or not there is an antibody that immunologically reacts with laminin or integrin in the sample, and the content of the antibody in the sample is measured. In other words, it is a concept that encompasses “quantitative”.
　抗ラミニン抗体検出工程及び抗インテグリン抗体検出工程は、検体中の前記抗体を定量的又は定性的に検出可能な方法であればよく、具体的な態様は特に限定されない。例えば、免疫学的手法を用いることができ、例えば、抗体酵素法（ＥＬＩＳＡ法）、免疫沈降法（ＩＰＰ法）、免疫ブロット法（ＩＢ法）、ラテックス凝集法、免疫クロマトグラフィー法、間接蛍光抗体法（ＩＦ法）、ラジオイムノアッセイ法（ＲＩＡ法）などの手法を挙げることができる。多くの検体を処理し得るＥＬＩＳＡ法が特に好ましい。ＥＬＩＳＡ法では、具体的には、前記抗体の抗原である、上記で詳述したようなラミニン又はインテグリンを固相に固定し、該固相に固定した抗原に検体を接触させ、抗原と、検体中に含有される可能性のある前記抗体との免疫複合体を検出することで、前記抗体を検出することができる。ＥＬＩＳＡ法の実施に伴う非特異反応の抑制方法や、検出の際に使用し得る標識物質、測定機器などは、特に限定されない。抗原を固定するための固相としては、プレート、ビーズ、チューブなどの任意の形状の固相を用いることができる。 The anti-laminin antibody detection step and the anti-integrin antibody detection step may be any method that can quantitatively or qualitatively detect the antibody in the specimen, and specific embodiments are not particularly limited. For example, immunological techniques can be used, for example, antibody enzyme method (ELISA method), immunoprecipitation method (IPP method), immunoblot method (IB method), latex agglutination method, immunochromatography method, indirect fluorescent antibody Methods (IF method), radioimmunoassay methods (RIA method) and the like can be mentioned. An ELISA method that can process many specimens is particularly preferred. In the ELISA method, specifically, laminin or integrin as described in detail above, which is the antigen of the antibody, is immobilized on a solid phase, the specimen is contacted with the antigen immobilized on the solid phase, and the antigen and specimen The antibody can be detected by detecting an immune complex with the antibody that may be contained therein. There are no particular limitations on the method for suppressing the non-specific reaction associated with the ELISA method, the labeling substance that can be used in the detection, the measuring instrument, and the like. As a solid phase for immobilizing an antigen, a solid phase having an arbitrary shape such as a plate, a bead, or a tube can be used.
　抗ラミニン抗体検出工程におけるラミニンと免疫学的に反応する抗体の量、及び、抗インテグリン抗体検出工程におけるインテグリンと免疫学的に反応する抗体の量は、前記抗体と前記抗原との免疫複合体の標識の測定値として求めることができる。既知濃度の前記抗体を含む標準試料により検量線を作成し、検量線を用いて標識の測定値から前記抗体の量を算出することもできる。 The amount of the antibody that immunologically reacts with laminin in the anti-laminin antibody detection step, and the amount of the antibody that immunologically reacts with integrin in the anti-integrin antibody detection step is the amount of the immune complex of the antibody and the antigen. It can be determined as a measured value of the label. It is also possible to prepare a calibration curve with a standard sample containing the antibody at a known concentration, and calculate the amount of the antibody from the measured value of the label using the calibration curve.
　抗ラミニン抗体検出工程におけるラミニンと免疫学的に反応する抗体が存在するか否かの判定（陽性／陰性の判定）、及び、抗インテグリン抗体検出工程におけるインテグリンと免疫学的に反応する抗体が存在するか否かの判定（陽性／陰性の判定）は、それぞれ、被検動物個体（例えば、ＩｇＧ４関連疾患の罹患が疑われるヒト）から得た検体中の前記抗体の測定値を、ＩｇＧ４関連疾患を罹患していない動物個体（例えば健常者）から得た検体中の前記抗体の測定値と比較することによって行うことができる。ここで、ＩｇＧ４関連疾患を罹患していない個体から得た検体中の前記抗体の測定値は、同時に測定して得た値であってもよいし、予め測定して得た値であってもよい。被検動物個体から得た検体中の前記抗体の測定値が、ＩｇＧ４関連疾患を罹患していない動物個体から得た検体中の前記抗体の測定値よりも有意に大きい場合に、陽性と判断することができる。 Determination of whether there is an antibody that immunologically reacts with laminin in the anti-laminin antibody detection process (positive / negative determination), and presence of an antibody that reacts immunologically with integrin in the anti-integrin antibody detection process The determination of whether or not to perform (positive / negative determination) is based on the measured value of the antibody in a specimen obtained from an individual animal subject (eg, a human suspected of suffering from an IgG4-related disease). Can be performed by comparing the measured value of the antibody in a sample obtained from an animal individual (for example, a healthy person) who does not suffer from the disease. Here, the measured value of the antibody in the sample obtained from an individual who does not suffer from an IgG4-related disease may be a value obtained by simultaneous measurement or may be a value obtained by measurement in advance. Good. When the measured value of the antibody in the specimen obtained from the test animal individual is significantly larger than the measured value of the antibody in the specimen obtained from the animal individual not suffering from IgG4-related disease, it is judged as positive be able to.
　抗ラミニン抗体検出工程におけるラミニンと免疫学的に反応する抗体、及び、抗インテグリン抗体検出工程におけるインテグリンと免疫学的に反応する抗体のクラスは特に限定されないが、好ましくはＩｇＧ抗体である。ＩｇＧ抗体のなかのサブクラスは特に限定されないが、例えばＩｇＧ１抗体又はＩｇＧ４抗体を検出することができる。本発明者らは、ＩｇＧ４関連疾患では、ラミニンと免疫学的に反応するＩｇＧ抗体、特にＩｇＧ１抗体又はＩｇＧ４抗体が病原物質であることを見出していることから、これらはＩｇＧ４関連疾患の指標として特に有用である。 The class of the antibody that immunologically reacts with laminin in the anti-laminin antibody detection step and the antibody that immunologically reacts with integrin in the anti-integrin antibody detection step are not particularly limited, but are preferably IgG antibodies. Although the subclass in IgG antibody is not specifically limited, For example, IgG1 antibody or IgG4 antibody can be detected. Since the present inventors have found that IgG antibodies immunologically reactive with laminin, in particular IgG1 antibodies or IgG4 antibodies, are pathogens in IgG4-related diseases, these are particularly useful as indicators of IgG4-related diseases. Useful.
　検査するＩｇＧ４関連疾患の種類と、ラミニンと免疫学的に反応する抗体の種類、又は、インテグリンと免疫学的に反応する抗体の種類との組み合わせは特に限定されない。 The combination of the type of IgG4-related disease to be examined, the type of antibody that immunologically reacts with laminin, or the type of antibody that immunologically reacts with integrin is not particularly limited.
　例えば、自己免疫性膵炎（ＡＩＰ）の指標としては、ラミニン－５１１と免疫学的に反応する抗体（実験４参照）及びインテグリンα６β１と免疫学的に反応する抗体（実験９参照）が有用である。ラミニン－５１１と免疫学的に反応する抗体は、ラミニン－５１１の断片（特に、インテグリン結合活性を有する３量体断片）を抗原として用いた検出法により前記抗体を検出することが特に好ましい。インテグリンα６β１と免疫学的に反応する抗体は、インテグリンα６β１を抗原として用いた検出法により前記抗体を検出することが特に好ましい。 For example, as an index of autoimmune pancreatitis (AIP), an antibody that immunologically reacts with laminin-511 (see Experiment 4) and an antibody that reacts immunologically with integrin α6β1 (see Experiment 9) are useful. . The antibody that immunologically reacts with laminin-511 is particularly preferably detected by a detection method using a fragment of laminin-511 (particularly, a trimer fragment having integrin binding activity) as an antigen. The antibody that reacts immunologically with integrin α6β1 is particularly preferably detected by a detection method using integrin α6β1 as an antigen.
　一方、ＩｇＧ４関連腎疾患の指標としては、ラミニン－５２１と免疫学的に反応する抗体が有用である（実験６参照）。この場合、ラミニン－５２１を抗原として用いた検出法により前記抗体を検出することが特に好ましい。 On the other hand, an antibody that immunologically reacts with laminin-521 is useful as an indicator of IgG4-related kidney disease (see Experiment 6). In this case, it is particularly preferable to detect the antibody by a detection method using laminin-521 as an antigen.
　本発明のＩｇＧ４関連疾患の検査方法の、抗ラミニン抗体検出工程を含む実施形態により、検体中の、ラミニン、特にラミニン－５１１、特にラミニン－５１１－Ｅ８と免疫学的に反応する抗体が検出された場合、検出結果に基づき、検体が由来する被験動物が、ＩｇＧ４関連疾患、特に自己免疫性膵炎（ＡＩＰ）に罹患していると判断することができる。ラミニン、特にラミニン－５１１、特にラミニン－５１１－Ｅ８と免疫学的に反応する抗体は、膵癌患者の検体からは検出されないことから、本発明のＩｇＧ４関連疾患の検査方法の、抗ラミニン抗体検出工程を含む実施形態は、ＡＩＰと膵癌とを判別する指標を得ることができ、特に有用である。 According to the embodiment of the method for testing an IgG4-related disease of the present invention including an anti-laminin antibody detection step, an antibody immunologically reactive with laminin, particularly laminin-511, particularly laminin-511-E8, is detected in a sample. In this case, based on the detection result, it can be determined that the test animal from which the specimen is derived suffers from an IgG4-related disease, particularly autoimmune pancreatitis (AIP). Since an antibody that immunologically reacts with laminin, particularly laminin-511, particularly laminin-511-E8, is not detected from a specimen of a pancreatic cancer patient, the anti-laminin antibody detection step of the IgG4-related disease testing method of the present invention In particular, the embodiment including the above can obtain an index for discriminating between AIP and pancreatic cancer, and is particularly useful.
　また、本発明のＩｇＧ４関連疾患の検査方法の、抗ラミニン抗体検出工程を含む実施形態により、検体中の、ラミニン、特にラミニン－５１１、特にラミニン－５１１－Ｅ８と免疫学的に反応する抗体が検出された場合、検出結果に基づき、検体が由来する被験動物が、ＩｇＧ４関連疾患、特にＡＩＰに、悪性腫瘍を合併するリスクが低いと判断することができる。 Further, according to the embodiment of the method for testing an IgG4-related disease of the present invention including an anti-laminin antibody detection step, an antibody that immunologically reacts with laminin, particularly laminin-511, particularly laminin-511-E8, in a sample. If detected, based on the detection result, it can be determined that the test animal from which the specimen is derived has a low risk of combining a malignant tumor with IgG4-related disease, particularly AIP.
　本発明のＩｇＧ４関連疾患の検査方法の、抗インテグリン抗体検出工程を含む実施形態により、検体中の、インテグリン、特にインテグリンα６β１と免疫学的に反応する抗体が検出された場合、検出結果に基づき、検体が由来する被験動物が、ＩｇＧ４関連疾患、特にＡＩＰに罹患していると判断することができる。 When an antibody that immunologically reacts with integrin, particularly integrin α6β1, is detected in the sample according to the embodiment of the method for testing an IgG4-related disease of the present invention including an anti-integrin antibody detection step, based on the detection result, It can be determined that the test animal from which the specimen is derived suffers from an IgG4-related disease, particularly AIP.
　膵癌を罹患する被験動物と、ＡＩＰを罹患する、抗インテグリン抗体陽性の被験動物とでは、膵画像が類似しており、膵画像所見のみによる区別は難しい。ところが、インテグリン、特にインテグリンα６β１と免疫学的に反応する抗体は、膵癌患者の検体からは検出されないことから、本発明のＩｇＧ４関連疾患の検査方法の、抗インテグリン抗体検出工程を含む実施形態は、ＡＩＰと膵癌とを判別する指標を得ることができるため特に有用である。 Pancreatic images are similar between test animals suffering from pancreatic cancer and anti-integrin antibody-positive test animals suffering from AIP, and it is difficult to distinguish only by pancreatic image findings. However, since an antibody that immunologically reacts with integrin, in particular with integrin α6β1, is not detected from a specimen of a patient with pancreatic cancer, an embodiment including the anti-integrin antibody detection step of the IgG4-related disease test method of the present invention comprises: This is particularly useful because an index for discriminating between AIP and pancreatic cancer can be obtained.
　また、本発明のＩｇＧ４関連疾患の検査方法の、抗インテグリン抗体検出工程を含む実施形態により、検体中に、インテグリン、特にインテグリンα６β１と免疫学的に反応する抗体が検出された場合、検出結果に基づき、検体が由来する被験動物が、ＩｇＧ４関連疾患、特にＡＩＰに、悪性腫瘍を合併するリスクが高いと判断することができる。悪性腫瘍を合併するリスクが高いと判断された被験動物に対しては、全身における悪性腫瘍のスクリーニングを実施する必要性が高いと判断することが可能である。 In addition, according to the embodiment of the method for testing an IgG4-related disease of the present invention including an anti-integrin antibody detection step, if an antibody immunologically reactive with integrin, particularly integrin α6β1, is detected in the sample, the detection result Based on this, it can be determined that the test animal from which the specimen is derived has a high risk of combining malignant tumors with IgG4-related diseases, particularly AIP. It is possible to determine that it is highly necessary to screen a malignant tumor throughout the body for a test animal that has been determined to have a high risk of complication of a malignant tumor.
　本発明のＩｇＧ４関連疾患の検査方法は、抗ラミニン抗体検出工程と抗インテグリン抗体検出工程の少なくとも一方を含んでいればよいが、より好ましくは、抗ラミニン抗体検出工程と抗インテグリン抗体検出工程の両方を含む。抗ラミニン抗体陰性の検体であっても、抗インテグリン抗体陽性である場合があり、抗インテグリン抗体陰性の検体であっても、抗ラミニン抗体陽性である場合があるため、検体中において両方の抗体を検出することにより、ＩｇＧ４関連疾患、特にＡＩＰ、に罹患している被験動物の検体の検出漏れの可能性を低減することができる。
＜３．ＩｇＧ４関連疾患の検査試薬＞
　本発明は第二に、
　ラミニンを含む、ＩｇＧ４関連疾患を検査するための検査試薬、
　インテグリンを含む、ＩｇＧ４関連疾患を検査するための検査試薬、或いは、
　ラミニン及びインテグリンを含む、ＩｇＧ４関連疾患を検査するための検査試薬
に関する。 The method for testing an IgG4-related disease of the present invention may include at least one of an anti-laminin antibody detection step and an anti-integrin antibody detection step, and more preferably both an anti-laminin antibody detection step and an anti-integrin antibody detection step. including. An anti-laminin antibody-negative sample may be anti-integrin antibody-positive, and an anti-integrin antibody-negative sample may be anti-laminin antibody-positive. By detecting, it is possible to reduce the possibility of detection failure of the specimen of the test animal suffering from IgG4-related disease, particularly AIP.
<3. Test reagent for IgG4-related disease>
The present invention secondly,
A test reagent for testing IgG4-related diseases, including laminin,
Test reagents for testing IgG4-related diseases, including integrins, or
The present invention relates to a test reagent for testing an IgG4-related disease, including laminin and integrin.
　ここで、ＩｇＧ４関連疾患、ラミニン、及び、インテグリンについては、本発明の検査方法に関して既述の通りである。 Here, IgG4-related diseases, laminin, and integrin are as described above for the test method of the present invention.
　本発明の検査試薬の、ラミニンを含む実施形態は、上記の本発明の検査方法の、抗ラミニン抗体検出工程を含む実施形態に使用することができる。 The embodiment of the test reagent of the present invention containing laminin can be used in the above-described test method of the present invention including an anti-laminin antibody detection step.
　本発明の検査試薬の、インテグリンを含む実施形態は、上記の本発明の検査方法の、抗インテグリン抗体検出工程を含む実施形態に使用することができる。 The embodiment of the test reagent of the present invention containing integrin can be used in the above-described test method of the present invention including an anti-integrin antibody detection step.
　本発明の検査試薬の１以上の実施形態は、免疫学的測定に必要な試薬類、例えば、緩衝液等を必要に応じて含んでよく、複数の成分からなるキットとして提供されてもよい。 One or more embodiments of the test reagent of the present invention may contain reagents necessary for immunological measurement, for example, a buffer solution as necessary, and may be provided as a kit comprising a plurality of components.
　本発明の検査試薬の、ラミニンを含む実施形態において、ラミニンの形態は特に限定されず、溶液状態であってもよいし、乾燥状態であってもよいし、固相に固定された形態であってもよい。ラミニンが乾燥状態である場合、使用前に溶液状態にするための緩衝液や溶媒を本発明の検査試薬に含めてもよい。 In the embodiment of the test reagent of the present invention containing laminin, the form of laminin is not particularly limited, and may be a solution state, a dry state, or a form fixed to a solid phase. May be. When laminin is in a dry state, a buffer solution or a solvent for making it into a solution state before use may be included in the test reagent of the present invention.
　本発明の検査試薬の、インテグリンを含む実施形態において、インテグリンの形態は特に限定されず、溶液状態であってもよいし、乾燥状態であってもよいし、固相に固定された形態であってもよい。インテグリンが乾燥状態である場合、使用前に溶液状態にするための緩衝液や溶媒を本発明の検査試薬に含めてもよい。 In the embodiment of the test reagent of the present invention containing integrin, the form of integrin is not particularly limited, and may be in a solution state, a dry state, or a form fixed on a solid phase. May be. When the integrin is in a dry state, a buffer solution or a solvent for making it into a solution state before use may be included in the test reagent of the present invention.
　固相に固定された形態のラミニンを含む本発明の検査試薬の実施形態は、ＥＬＩＳＡ法によりＩｇＧ４関連疾患を検査するためのキットの形態とすることができる。固相に固定された形態のインテグリンを含む本発明の検査試薬の実施形態もまた、ＥＬＩＳＡ法によりＩｇＧ４関連疾患を検査するためのキットの形態とすることができる。固相に固定された形態のラミニン及びインテグリンを含む本発明の検査試薬の実施形態もまた、ＥＬＩＳＡ法によりＩｇＧ４関連疾患を検査するためのキットの形態とすることができる。各実施形態において、固相の形態としては、プレート、ビーズ、チューブなどの任意の形態であってよい。ＥＬＩＳＡ法によりＩｇＧ４関連疾患を検査するためのキットは、前記固相以外に、ブロッキング液、洗浄液、検体希釈液、酵素標識した二次抗体、基質液等を含むことができる。
＜４．ＩｇＧ４関連疾患に対する治療の効果を評価する方法＞
　本発明は第三に、
　ＩｇＧ４関連疾患に対する治療の効果を評価する方法であって、
　ＩｇＧ４関連疾患に対する治療が施された被検動物から得た検体中の、ラミニンと免疫学的に反応する抗体を検出する抗ラミニン抗体検出工程、及び／又は、
　ＩｇＧ４関連疾患に対する治療が施された被検動物から得た検体中の、インテグリンと免疫学的に反応する抗体を検出する抗インテグリン抗体検出工程
を含む方法に関する。 An embodiment of the test reagent of the present invention containing laminin in a form immobilized on a solid phase can be in the form of a kit for testing an IgG4-related disease by ELISA. An embodiment of the test reagent of the present invention containing the integrin in a form immobilized on a solid phase can also be in the form of a kit for testing an IgG4-related disease by ELISA. An embodiment of the test reagent of the present invention comprising laminin and integrin in a form immobilized on a solid phase can also be in the form of a kit for testing IgG4-related diseases by ELISA. In each embodiment, the form of the solid phase may be any form such as a plate, a bead, or a tube. In addition to the solid phase, a kit for examining an IgG4-related disease by ELISA can contain a blocking solution, a washing solution, a sample diluent, an enzyme-labeled secondary antibody, a substrate solution, and the like.
<4. Method for evaluating the effect of treatment on IgG4-related diseases>
Third, the present invention
A method for assessing the effect of treatment on an IgG4-related disease comprising:
An anti-laminin antibody detection step of detecting an antibody that immunologically reacts with laminin in a specimen obtained from a test animal treated for an IgG4-related disease; and / or
The present invention relates to a method comprising an anti-integrin antibody detection step of detecting an antibody that immunologically reacts with an integrin in a specimen obtained from a test animal treated for an IgG4-related disease.
　上記の「ＩｇＧ４関連疾患に対する治療の効果を評価する方法」は、「ＩｇＧ４関連疾患に対する治療の効果を評価するための指標を取得する方法」と表現してもよい。 The above-mentioned “method for evaluating the effect of treatment on an IgG4-related disease” may be expressed as “a method for obtaining an index for evaluating the effect of treatment on an IgG4-related disease”.
　本発明の検査方法に関して既述の通り、検体中の、ラミニンと免疫学的に反応する抗体、及び、インテグリンと免疫学的に反応する抗体は、ＩｇＧ４関連疾患の指標として有用である。このため、ＩｇＧ４関連疾患に対する治療が施された被検動物から得た検体中の前記抗体は、治療が有効で治癒している場合は低下し、治療が十分に効果を発揮していない場合は治療開始前から変化しない又は上昇する。そこで、ＩｇＧ４関連疾患に対する治療が施された被検動物から得た検体中の。ラミニンと免疫学的に反応する抗体、及び／又は、インテグリンと免疫学的に反応する抗体を検出することで、治療の効果を評価することが可能となる。 As described above with regard to the test method of the present invention, the antibody that reacts immunologically with laminin and the antibody that reacts immunologically with integrin in the specimen are useful as indicators of IgG4-related diseases. For this reason, the antibody in the specimen obtained from the test animal treated for IgG4-related disease is reduced when the treatment is effective and cured, and when the treatment is not sufficiently effective It remains unchanged or rises from before the start of treatment. Thus, in a sample obtained from a test animal treated for IgG4-related disease. By detecting an antibody immunologically reactive with laminin and / or an antibody immunologically reactive with integrin, it is possible to evaluate the effect of treatment.
　被検動物としては、ＩｇＧ４関連疾患の治療を受けたヒトや非ヒト動物が例示できる。 Examples of test animals include humans and non-human animals that have been treated for IgG4-related diseases.
　ＩｇＧ４関連疾患の治療としては、ステロイド投与等の、ＩｇＧ４関連疾患の治療が挙げられる。 Treatment of IgG4-related diseases includes treatment of IgG4-related diseases such as steroid administration.
　本発明の治療効果の評価方法における、検体、ラミニン、インテグリン、抗体、抗ラミニン抗体検出工程、抗インテグリン抗体検出工程などの具体的態様は、本発明の検査方法に関して説明したのと同様である。 Specific examples of the method for evaluating the therapeutic effect of the present invention, such as the specimen, laminin, integrin, antibody, anti-laminin antibody detection step, anti-integrin antibody detection step, etc., are the same as described for the test method of the present invention.
　本発明の治療効果の評価方法における抗ラミニン抗体検出工程及び／又は抗インテグリン抗体検出工程で得た、ＩｇＧ４関連疾患に対する治療が施された被検動物から得た検体中のラミニンと免疫学的に反応する抗体及び／又はインテグリンと免疫学的に反応する抗体の定量結果を、例えば、前記治療を開始する前の同一動物個体から得た検体中のラミニンと免疫学的に反応する抗体及び／又はインテグリンと免疫学的に反応する抗体の定量結果と比較して、前者が後者よりも小さい場合には、治療が有効であると評価することができる。一方、前者が後者と同程度或いは後者よりも大きい場合には、治療が有効でない又は十分でないと評価することができる。この評価結果に基づいて、治療を更に継続する、治療方針を変更する、治療を中止するなどの判断が可能である。 Laminin in a specimen obtained from a test animal treated with an IgG4-related disease obtained in the anti-laminin antibody detection step and / or anti-integrin antibody detection step in the method for evaluating therapeutic effect of the present invention immunologically Quantitative results of antibodies that react immunologically with reacting antibodies and / or integrins, for example, antibodies that react immunologically with laminin in a specimen obtained from the same animal individual prior to the start of the treatment and / or When the former is smaller than the latter compared with the quantitative result of the antibody that immunologically reacts with integrin, it can be evaluated that the treatment is effective. On the other hand, if the former is about the same as or larger than the latter, it can be evaluated that the treatment is not effective or sufficient. Based on the evaluation result, it is possible to determine whether to continue the treatment, change the treatment policy, or stop the treatment.
　また、本発明の治療効果の評価方法における抗ラミニン抗体検出工程及び／又は抗インテグリン抗体検出工程で得た、ＩｇＧ４関連疾患に対する治療が施された被検動物から得た検体中のラミニンと免疫学的に反応する抗体及び／又はインテグリンと免疫学的に反応する抗体の定量結果を、例えば、同一動物種の健常個体から得た検体中のラミニンと免疫学的に反応する抗体及び／又はインテグリンと免疫学的に反応する抗体の定量結果と比較して、前者が後者と同程度である又はより小さい場合には、治療が有効であると評価することができる。一方、前者が後者よりも大きい場合には、治療が有効でない又は十分でないと評価することができる。この評価結果に基づいて、治療を更に継続する、治療方針を変更する、治療を中止するなどの判断が可能である。
＜５．ＩｇＧ４関連疾患の治療薬の候補物質をスクリーニングする方法１＞
　本発明は第四に、
　ＩｇＧ４関連疾患の治療薬の候補物質をスクリーニングする方法であって、
　試験物質を作用させた動物から得た検体中の、ラミニンと免疫学的に反応する抗体を検出する抗ラミニン抗体検出工程と、
　前記試験物質を作用させたことにより前記検体中の前記抗体が減少した場合に、前記試験物質を、ＩｇＧ４関連疾患の治療薬の候補物質として選抜する選抜工程と
を含む方法に関する。 Further, laminin and immunology in a specimen obtained from a test animal treated with an IgG4-related disease obtained in the anti-laminin antibody detection step and / or anti-integrin antibody detection step in the method for evaluating therapeutic effect of the present invention Quantification results of antibodies that react immunologically and / or antibodies that react immunologically with integrins, for example, antibodies and / or integrins that react immunologically with laminin in specimens obtained from healthy individuals of the same animal species If the former is comparable or smaller than the latter compared to the quantification result of the immunologically reactive antibody, it can be evaluated that the treatment is effective. On the other hand, if the former is larger than the latter, it can be evaluated that the treatment is not effective or sufficient. Based on the evaluation result, it is possible to determine whether to continue the treatment, change the treatment policy, or stop the treatment.
<5. Method 1 for Screening Candidate Substances for Treatment of IgG4-Related Disease>
Fourth, the present invention
A method for screening candidate substances for therapeutic agents for IgG4-related diseases, comprising:
An anti-laminin antibody detection step for detecting an antibody that immunologically reacts with laminin in a specimen obtained from an animal on which a test substance has been allowed to act;
And a selection step of selecting the test substance as a candidate substance for a therapeutic drug for IgG4-related disease when the antibody in the specimen decreases due to the action of the test substance.
　本発明の検査方法の抗ラミニン抗体検出工程を含む実施形態に関して既述の通り、検体中の、ラミニンと免疫学的に反応する抗体は、ＩｇＧ４関連疾患の指標として有用である。このため、試験物質を作用させたことにより前記検体中の前記抗体が減少した場合に、前記試験物質を、ＩｇＧ４関連疾患の治療薬の候補物質として選抜することが可能となる。 As described above with respect to the embodiment including the anti-laminin antibody detection step of the test method of the present invention, the antibody that immunologically reacts with laminin in the sample is useful as an indicator of IgG4-related diseases. For this reason, when the antibody in the sample decreases due to the action of a test substance, the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases.
　本発明のスクリーニング方法の抗ラミニン抗体検出工程を含む実施形態における、検体、ラミニン、抗体、抗ラミニン抗体検出工程などの具体的態様は、本発明の検査方法の抗ラミニン抗体検出工程を含む実施形態に関して説明したのと同様である。 In the embodiment including the anti-laminin antibody detection step of the screening method of the present invention, specific embodiments such as the specimen, laminin, antibody, and anti-laminin antibody detection step are embodiments including the anti-laminin antibody detection step of the test method of the present invention. This is the same as described above.
　本発明のスクリーニング方法の抗ラミニン抗体検出工程を含む実施形態において、試験物質としては、新規医薬の候補となる可能性のある試験物質であり特に限定されない。試験物質を作用させる動物は、典型的には、ＩｇＧ４関連疾患の非ヒトモデル動物等の、健常個体と比較して、ラミニンと免疫学的に反応する抗体の検体中での量が大きい動物である。 In the embodiment including the anti-laminin antibody detection step of the screening method of the present invention, the test substance is a test substance that may be a candidate for a new drug, and is not particularly limited. The animal to which the test substance is allowed to act is typically an animal having a large amount of an antibody that immunologically reacts with laminin in a specimen, such as a non-human animal model of an IgG4-related disease. is there.
　本発明のスクリーニング方法の抗ラミニン抗体検出工程を含む実施形態における選抜工程では、抗ラミニン抗体検出工程で得た、試験物質を作用させた動物から得た検体中の前記抗体の定量結果を、例えば、前記試験物質を作用させる前の同一動物個体から得た検体中の前記抗体の定量結果と比較して、前者が後者よりも小さい場合には、前記試験物質を作用させたことにより前記検体中の前記抗体が減少したと判断し、前記試験物質を、ＩｇＧ４関連疾患の治療薬の候補物質として選抜することができる。 In the selection step in the embodiment including the anti-laminin antibody detection step of the screening method of the present invention, the quantification result of the antibody in the specimen obtained from the animal to which the test substance is applied, obtained in the anti-laminin antibody detection step, for example, When the former is smaller than the latter compared with the quantification result of the antibody in the specimen obtained from the same animal individual before the test substance is allowed to act, Thus, the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases.
　また、本発明のスクリーニング方法の抗ラミニン抗体検出工程を含む実施形態における選抜工程では、抗ラミニン抗体検出工程で得た、試験物質を作用させた動物から得た検体中の前記抗体の定量結果を、例えば、同一動物種の健常個体から得た検体中の前記抗体の定量結果と比較して、前者が後者と同程度である又はより小さい場合には、前記試験物質を作用させたことにより前記検体中の前記抗体が減少したと判断し、前記試験物質を、ＩｇＧ４関連疾患の治療薬の候補物質として選抜することができる。
＜６．ＩｇＧ４関連疾患の治療薬の候補物質をスクリーニングする方法２＞
　本発明は第五に、
　ＩｇＧ４関連疾患の治療薬の候補物質をスクリーニングする方法であって、
　試験物質を作用させた動物から得た検体中の、インテグリンと免疫学的に反応する抗体を検出する抗インテグリン抗体検出工程と、
　前記試験物質を作用させたことにより前記検体中の前記抗体が減少した場合に、前記試験物質を、ＩｇＧ４関連疾患の治療薬の候補物質として選抜する選抜工程と
を含む方法に関する。 Further, in the selection step in the embodiment including the anti-laminin antibody detection step of the screening method of the present invention, the quantification result of the antibody in the specimen obtained from the animal to which the test substance is applied, obtained in the anti-laminin antibody detection step, is obtained. For example, when the former is comparable or smaller than the latter in comparison with the quantification result of the antibody in a sample obtained from a healthy individual of the same animal species, Judging that the antibody in the sample has decreased, the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases.
<6. Method 2 for Screening Candidate Substance for Treatment of IgG4-Related Disease>
Fifth, the present invention
A method for screening candidate substances for therapeutic agents for IgG4-related diseases, comprising:
An anti-integrin antibody detection step for detecting an antibody that immunologically reacts with an integrin in a sample obtained from an animal on which a test substance has been allowed to act;
And a selection step of selecting the test substance as a candidate substance for a therapeutic drug for IgG4-related disease when the antibody in the specimen decreases due to the action of the test substance.
　本発明の検査方法の抗インテグリン抗体検出工程を含む実施形態に関して既述の通り、検体中の、インテグリンと免疫学的に反応する抗体は、ＩｇＧ４関連疾患の指標として有用である。このため、試験物質を作用させたことにより前記検体中の前記抗体が減少した場合に、前記試験物質を、ＩｇＧ４関連疾患の治療薬の候補物質として選抜することが可能となる。 As described above with respect to the embodiment including the anti-integrin antibody detection step of the test method of the present invention, the antibody that immunologically reacts with the integrin in the sample is useful as an indicator of IgG4-related diseases. For this reason, when the antibody in the sample decreases due to the action of a test substance, the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases.
　本発明のスクリーニング方法の抗インテグリン抗体検出工程を含む実施形態における、検体、インテグリン、抗体、抗インテグリン抗体検出工程などの具体的態様は、本発明の検査方法の抗インテグリン抗体検出工程を含む実施形態に関して説明したのと同様である。 In the embodiment including the anti-integrin antibody detection step of the screening method of the present invention, specific embodiments such as the specimen, integrin, antibody, and anti-integrin antibody detection step are embodiments including the anti-integrin antibody detection step of the test method of the present invention. This is the same as described above.
　本発明のスクリーニング方法の抗インテグリン抗体検出工程を含む実施形態において、試験物質としては、新規医薬の候補となる可能性のある試験物質であり特に限定されない。試験物質を作用させる動物は、典型的には、ＩｇＧ４関連疾患の非ヒトモデル動物等の、健常個体と比較して、インテグリンと免疫学的に反応する抗体の検体中での量が大きい動物である。 In the embodiment including the anti-integrin antibody detection step of the screening method of the present invention, the test substance is not particularly limited and is a test substance that may be a candidate for a new drug. The animal to which the test substance is allowed to act is typically an animal having a large amount of an antibody that immunologically reacts with integrin compared to a healthy individual, such as a non-human animal model of an IgG4-related disease. is there.
　本発明のスクリーニング方法の抗インテグリン抗体検出工程を含む実施形態における選抜工程では、抗インテグリン抗体検出工程で得た、試験物質を作用させた動物から得た検体中の前記抗体の定量結果を、例えば、前記試験物質を作用させる前の同一動物個体から得た検体中の前記抗体の定量結果と比較して、前者が後者よりも小さい場合には、前記試験物質を作用させたことにより前記検体中の前記抗体が減少したと判断し、前記試験物質を、ＩｇＧ４関連疾患の治療薬の候補物質として選抜することができる。 In the selection step in the embodiment including the anti-integrin antibody detection step of the screening method of the present invention, the quantification result of the antibody in the specimen obtained from the animal to which the test substance is applied, obtained in the anti-integrin antibody detection step, for example, When the former is smaller than the latter compared with the quantification result of the antibody in the specimen obtained from the same animal individual before the test substance is allowed to act, Thus, the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases.
　また、本発明のスクリーニング方法の抗インテグリン抗体検出工程を含む実施形態における選抜工程では、抗インテグリン抗体検出工程で得た、試験物質を作用させた動物から得た検体中の前記抗体の定量結果を、例えば、同一動物種の健常個体から得た検体中の前記抗体の定量結果と比較して、前者が後者と同程度である又はより小さい場合には、前記試験物質を作用させたことにより前記検体中の前記抗体が減少したと判断し、前記試験物質を、ＩｇＧ４関連疾患の治療薬の候補物質として選抜することができる。
＜７．ＩｇＧ４関連疾患の非ヒトモデル動物を作製する方法１＞
　本発明は第六に、
　ＩｇＧ４関連疾患の非ヒトモデル動物を作製する方法であって、
　非ヒト動物に、ラミニンと免疫学的に反応する抗体を投与する抗ラミニン抗体投与工程、及び
　非ヒト動物を、ラミニンを抗原として免疫するラミニン免疫工程
のうち少なくとも一方を含む方法に関する。 Further, in the selection step in the embodiment including the anti-integrin antibody detection step of the screening method of the present invention, the quantification result of the antibody in the specimen obtained from the animal to which the test substance is applied, obtained in the anti-integrin antibody detection step, is obtained. For example, when the former is comparable or smaller than the latter in comparison with the quantification result of the antibody in a sample obtained from a healthy individual of the same animal species, Judging that the antibody in the sample has decreased, the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases.
<7. Method 1 for producing non-human animal model of IgG4-related disease>
Sixth, the present invention
A method for producing a non-human animal model of an IgG4-related disease comprising:
The present invention relates to a method comprising at least one of an anti-laminin antibody administration step of administering an antibody that immunologically reacts with laminin to a non-human animal, and a laminin immunization step of immunizing a non-human animal with laminin as an antigen.
　本発明の非ヒトモデル動物の作製方法の抗ラミニン抗体投与工程又はラミニン免疫工程を含む実施形態での、ラミニン、抗体等の具体的形態は、本発明の検査方法の抗ラミニン抗体検出工程を含む実施形態におけるものと同様である。 Specific embodiments of laminin, antibody, etc. in the embodiment including the anti-laminin antibody administration step or laminin immunization step of the method for producing a non-human model animal of the present invention include the anti-laminin antibody detection step of the test method of the present invention. This is the same as in the embodiment.
　本明細書に記載の実験１及び実験２では、ＩｇＧ４関連疾患患者から得た血清又は該血清から精製した抗体をマウスに投与したとき、マウスはＩｇＧ４関連疾患を発症することが確認されている。そして、実験４では、ＩｇＧ４関連疾患患者から得た血清が、ラミニンと免疫学的に反応する抗体を含むことが確認されている。このことから、非ヒト動物に、ラミニンと免疫学的に反応する抗体を投与する抗ラミニン抗体投与工程により、ＩｇＧ４関連疾患の非ヒトモデル動物の作製が可能であると言える。 In Experiment 1 and Experiment 2 described in this specification, it has been confirmed that when a serum obtained from an IgG4-related disease patient or an antibody purified from the serum is administered to the mouse, the mouse develops an IgG4-related disease. In Experiment 4, it has been confirmed that serum obtained from a patient with an IgG4-related disease contains an antibody that immunologically reacts with laminin. From this, it can be said that a non-human model animal of an IgG4-related disease can be produced by an anti-laminin antibody administration step in which an antibody that immunologically reacts with laminin is administered to the non-human animal.
　抗ラミニン抗体投与工程における非ヒト動物としては、例えばＢＡＬＢ／ｃマウス、Ｂ６マウス等のマウス、その他の非ヒト動物が例示できる。 Examples of non-human animals in the anti-laminin antibody administration step include mice such as BALB / c mice and B6 mice, and other non-human animals.
　抗ラミニン抗体投与工程における、ラミニンと免疫学的に反応する抗体の投与経路は特に限定されないが、例えば、皮下投与、腹腔内投与、静脈投与等が例示できる。ラミニンと免疫学的に反応する抗体は、精製された抗体として非ヒト動物に投与される必要はなく、例えば、抗体を含む血清として非ヒト動物に投与されてもよい。 In the anti-laminin antibody administration step, the administration route of the antibody that immunologically reacts with laminin is not particularly limited, and examples thereof include subcutaneous administration, intraperitoneal administration, and intravenous administration. An antibody that immunologically reacts with laminin need not be administered to a non-human animal as a purified antibody, and may be administered to a non-human animal as serum containing the antibody, for example.
　抗ラミニン抗体投与工程において、ラミニンと免疫学的に反応する抗体は、投与を受ける非ヒト動物が有するラミニンと免疫学的に反応できるように選択する。例えば、ヒト由来抗ヒトラミニン抗体は、マウスが有するラミニンと免疫学的に反応することができ、マウスにおいてＩｇＧ４関連疾患の病変を惹起することができる。 In the anti-laminin antibody administration step, the antibody that immunologically reacts with laminin is selected so that it can immunologically react with laminin of the non-human animal to be administered. For example, a human-derived anti-human laminin antibody can immunologically react with laminin possessed by mice, and can cause lesions of IgG4-related diseases in mice.
　一方、本明細書に記載の実験５では、ヒトラミニン－５１１－Ｅ８フラグメントを抗原としマウスを免疫したとき、該抗原に対する抗体が形成され、該マウスはＩｇＧ４関連疾患を発症することが確認されている。このことから、非ヒト動物を、ラミニンを抗原として免疫するラミニン免疫工程により、ＩｇＧ４関連疾患の非ヒトモデル動物の作製が可能であると言える。 On the other hand, in Experiment 5 described herein, when a mouse was immunized with human laminin-511-E8 fragment as an antigen, it was confirmed that an antibody against the antigen was formed and that the mouse developed an IgG4-related disease. . From this, it can be said that a non-human animal model of an IgG4-related disease can be produced by a laminin immunization process in which a non-human animal is immunized using laminin as an antigen.
　ラミニン免疫工程における非ヒト動物としては、例えばＢＡＬＢ／ｃマウス、Ｂ６マウス等のマウス、その他の非ヒト動物が例示できる。 Examples of non-human animals in the laminin immunization process include mice such as BALB / c mice and B6 mice, and other non-human animals.
　ラミニン免疫工程における方法は特に限定されない。ラミニンを、完全フロイントアジュバント等の適当なアジュバントとともに、非ヒト動物に皮下投与、静脈内投与、或いは腹腔内投与等の経路で投与することで、非ヒト動物を免疫することができる。 The method in the laminin immunization process is not particularly limited. A non-human animal can be immunized by administering laminin together with a suitable adjuvant such as complete Freund's adjuvant to the non-human animal by a route such as subcutaneous administration, intravenous administration, or intraperitoneal administration.
　ラミニン免疫工程において、抗原として投与するラミニンは、それに対する抗体が、免疫される非ヒト動物が有するラミニンに自己免疫により免疫学的に反応できるように選択する。例えば、ヒトラミニン－５１１は、それに対するマウス抗体が、マウスが有するラミニンと自己免疫により免疫学的に反応することができ、マウスにおいてＩｇＧ４関連疾患の病変を惹起することができる。
＜８．ＩｇＧ４関連疾患の非ヒトモデル動物を作製する方法２＞
　本発明は第七に、
　ＩｇＧ４関連疾患の非ヒトモデル動物を作製する方法であって、
　非ヒト動物に、インテグリンと免疫学的に反応する抗体を投与する抗インテグリン抗体投与工程、及び
　非ヒト動物を、インテグリンを抗原として免疫するインテグリン免疫工程
のうち少なくとも一方を含む方法に関する。 In the laminin immunization step, laminin to be administered as an antigen is selected so that an antibody against it can immunologically respond to laminin possessed by the non-human animal to be immunized by autoimmunity. For example, human laminin-511 can react immunologically with mouse antibodies against mouse laminin, and can cause lesions of IgG4-related diseases in mice.
<8. Method 2 for producing non-human animal model of IgG4-related disease>
Seventh, the present invention
A method for producing a non-human animal model of an IgG4-related disease comprising:
The present invention relates to a method comprising at least one of an anti-integrin antibody administration step of administering an antibody that immunologically reacts with an integrin to a non-human animal, and an integrin immunization step of immunizing the non-human animal with an integrin as an antigen.
　本発明の非ヒトモデル動物の作製方法の抗インテグリン抗体投与工程又はインテグリン免疫工程を含む実施形態での、インテグリン、抗体等の具体的形態は、本発明の検査方法の抗インテグリン抗体検出工程を含む実施形態におけるものと同様である。 In the embodiment including the anti-integrin antibody administration step or the integrin immunization step of the method for producing a non-human model animal of the present invention, the specific form of integrin, antibody, etc. includes the anti-integrin antibody detection step of the test method of the present invention. This is the same as in the embodiment.
　抗インテグリン抗体投与工程における非ヒト動物としては、例えばＢＡＬＢ／ｃマウス、Ｂ６マウス等のマウス、その他の非ヒト動物が例示できる。 Examples of non-human animals in the anti-integrin antibody administration step include mice such as BALB / c mice and B6 mice, and other non-human animals.
　抗インテグリン抗体投与工程における、インテグリンと免疫学的に反応する抗体の投与経路は特に限定されないが、例えば、皮下投与、腹腔内投与、静脈投与等が例示できる。インテグリンと免疫学的に反応する抗体は、精製された抗体として非ヒト動物に投与される必要はなく、例えば、抗体を含む血清として非ヒト動物に投与されてもよい。 The administration route of the antibody that immunologically reacts with the integrin in the anti-integrin antibody administration step is not particularly limited, and examples thereof include subcutaneous administration, intraperitoneal administration, and intravenous administration. The antibody that immunologically reacts with the integrin need not be administered to a non-human animal as a purified antibody, and may be administered to a non-human animal as serum containing the antibody, for example.
　抗インテグリン抗体投与工程において、インテグリンと免疫学的に反応する抗体は、投与を受ける非ヒト動物が有するインテグリンと免疫学的に反応できるように選択する。例えば、ヒト由来抗ヒトインテグリン抗体（特に、ヒト由来抗ヒトインテグリンα６β１抗体）は、マウスが有するインテグリンと免疫学的に反応することができ、マウスにおいてＩｇＧ４関連疾患の病変を惹起することができる。 In the anti-integrin antibody administration step, the antibody that immunologically reacts with the integrin is selected so that it can react immunologically with the integrin of the non-human animal to be administered. For example, a human-derived anti-human integrin antibody (particularly, a human-derived anti-human integrin α6β1 antibody) can immunologically react with an integrin possessed by a mouse, and can cause a lesion of an IgG4-related disease in the mouse.
　インテグリン免疫工程における非ヒト動物としては、例えばＢＡＬＢ／ｃマウス、Ｂ６マウス等のマウス、その他の非ヒト動物が例示できる。 Examples of non-human animals in the integrin immunization step include mice such as BALB / c mice and B6 mice, and other non-human animals.
　インテグリン免疫工程における方法は特に限定されない。インテグリンを、完全フロイントアジュバント等の適当なアジュバントとともに、非ヒト動物に皮下投与、静脈内投与、或いは腹腔内投与等の経路で投与することで、非ヒト動物を免疫することができる。 The method in the integrin immunization process is not particularly limited. A non-human animal can be immunized by administering the integrin together with a suitable adjuvant such as complete Freund's adjuvant to the non-human animal by a route such as subcutaneous administration, intravenous administration, or intraperitoneal administration.
　インテグリン免疫工程において、抗原として投与するインテグリンは、それに対する抗体が、免疫される非ヒト動物が有するインテグリンに自己免疫により免疫学的に反応できるように選択する。例えば、ヒトインテグリン（特にヒトインテグリンα６β１）は、それに対するマウス抗体が、マウスが有するインテグリンと自己免疫により免疫学的に反応することができ、マウスにおいてＩｇＧ４関連疾患の病変を惹起することができる。 In the integrin immunization step, the integrin to be administered as an antigen is selected so that the antibody against it can react immunologically with the integrin of the non-human animal to be immunized by autoimmunity. For example, human integrins (particularly human integrin α6β1) can react immunologically with mouse integrins by autoimmunity with mouse antibodies against them, and can cause lesions of IgG4-related diseases in mice.
＜１．実験１：ＩｇＧ４関連疾患患者のＩｇＧのマウスへの投与＞
　実験１では、ＩｇＧ４関連疾患患者の血清から精製したＩｇＧを健常マウス新生仔に投与し、投与後の膵臓への影響を観察した。その結果、ＩｇＧ４関連疾患患者の血清から精製したＩｇＧは、膵臓に対して病原性を示すことが明らかとなった。
１．１．血清
　ＩｇＧ４関連疾患と診断された１０名の患者から血清試料を取得した。 <1. Experiment 1: IgG administration of IgG4-related disease patients to mice>
In Experiment 1, IgG purified from the serum of an IgG4-related disease patient was administered to healthy newborn mice, and the effect on the pancreas after administration was observed. As a result, it was revealed that IgG purified from the serum of patients with IgG4-related diseases showed pathogenicity to the pancreas.
1.1. Serum serum samples were obtained from 10 patients diagnosed with IgG4-related disease.
　対照血清試料として、５名の健常者からの血清試料と、５名の疾患患者（３名の膵癌患者、２名の原発性硬化性胆管炎患者）からの血清試料を取得した。対照血清試料は、ＩｇＧ４関連疾患患者血清試料と、年齢（±５歳）、性別を揃えた。
１．２．ヒトＩｇＧの調製
　上記の血清試料から、Ａｂ－Ｒａｐｉｄ　ＰｕＲｅ　Ｅｘ（Ｐ－０１５，Ｐｏｒｔｅ　Ｎｏｖａ製）を用い、製品の指示書に沿った手順により、ヒトＩｇＧを調製した。ＩｇＧはｐＨ７．２のリン酸緩衝生理食塩水（ＰＢＳ）により透析し、Ａｍｉｃｏｎ　Ｕｌｔｒａ（ＵＦＣ８０５０２４，Ｍｉｌｌｉｐｏｒｅ製）を用いた限外濾過により濃縮し、使用時まで－２０℃で保存した。精製されたＩｇＧの濃度は、Ｈｕｍａｎ　ＩｇＧ　ＥＩＡ　Ｋｉｔ（ＭＫ１３６，Ｔａｋａｒａ製）を用いて測定した。ＩｇＧ画分の純度を、Ｈｕｍａｎ　ＩｇＡ　ＥＬＩＳＡ　Ｋｉｔ（Ｅ８８－１０２，　Ｂｅｔｈｙｌ　Ｌａｂｏｒａｔｏｒｉｒｅｓ製）、Ｈｕｍａｎ　ＩｇＭ　ＥＬＩＳＡ　Ｋｉｔ（Ｅ８８－１００，　Ｂｅｔｈｙｌ　Ｌａｂｏｒａｔｏｒｉｒｅｓ製）、Ｈｕｍａｎ　ＩｇＥ　ＥＬＩＳＡ　Ｋｉｔ（Ｅ８８－１０８，　Ｂｅｔｈｙｌ　Ｌａｂｏｒａｔｏｒｉｒｅｓ製）、及び、クーマシーブリリアントブルー染色をそれぞれ用いてＩｇＡ、ＩｇＭ、ＩｇＥ及びタンパク質の混入を求め、確認した。 As control serum samples, serum samples from 5 healthy subjects and serum samples from 5 disease patients (3 pancreatic cancer patients, 2 primary sclerosing cholangitis patients) were obtained. The control serum sample had the same age (± 5 years) and sex as the serum sample of an IgG4-related disease patient.
1.2. Preparation of human IgG Human IgG was prepared from the above serum sample using Ab-Rapid PuRe Ex (manufactured by P-015, Porte Nova) according to the procedure according to the product instructions. IgG was dialyzed against phosphate buffered saline (PBS) at pH 7.2, concentrated by ultrafiltration using Amicon Ultra (UFC805024, manufactured by Millipore), and stored at −20 ° C. until use. The concentration of purified IgG was measured using a Human IgG EIA Kit (MK136, manufactured by Takara). The purity of the IgG fraction was determined using Human IgA ELISA Kit (E88-102, manufactured by Ethyl Laboratories), Human IgM ELISA Kit (manufactured by E88-100, manufactured by Ethyl Laboratores), Human IgE ELISA 108-L (manufactured by Human IgE ELISA-Lit). The contamination of IgA, IgM, IgE and protein was determined and confirmed using Coomassie brilliant blue staining, respectively.
　ＩｇＧ４関連疾患患者の血清試料から得られたＩｇＧを「患者ＩｇＧ」とした。対照血清試料から得られたＩｇＧを「対照ＩｇＧ」とした。
１．３．動物試験
　マウスとしてＢＡＬＢ／ｃマウスの雄の新生仔を用いた。 IgG obtained from a serum sample of an IgG4-related disease patient was designated as “patient IgG”. The IgG obtained from the control serum sample was designated as “control IgG”.
1.3. Animal test Male newborns of BALB / c mice were used as mice.
　各血清試料から調製されたＩｇＧをマウスに皮下投与した。１０～２０ｍｇのヒトＩｇＧをマウス体重１ｇに対して投与した。
１．４．結果と考察１（図１Ａ～図１Ｃ）
　患者ＩｇＧ又は対照ＩｇＧの皮下投与から１２時間後に、マウスの膵臓の組織切片を採取し、Ｈ＆Ｅ（ヘマトキシリン＆エオシン）染色及び免疫組織化学的染色により染色し、観察した。免疫組織化学的染色では、骨髄細胞分化抗原Ｇｒ１に対する抗体を一次抗体として用い、Ｇｒ１陽性細胞を染色した。 IgG prepared from each serum sample was administered subcutaneously to mice. 10-20 mg of human IgG was administered to 1 g of mouse body weight.
1.4. Results and discussion 1 (FIGS. 1A to 1C)
Twelve hours after subcutaneous administration of patient IgG or control IgG, mouse pancreas tissue sections were collected, stained by H & E (hematoxylin & eosin) staining and immunohistochemical staining, and observed. In immunohistochemical staining, Gr1-positive cells were stained using an antibody against bone marrow cell differentiation antigen Gr1 as a primary antibody.
　また、皮下投与から１２時間後に、マウスの血清中のヒトＩｇＧ濃度を測定した。 Moreover, the human IgG concentration in the mouse serum was measured 12 hours after subcutaneous administration.
　結果を図１Ａ～図１Ｃに示す。 The results are shown in FIGS. 1A to 1C.
　図１Ａは、対照ＩｇＧ投与マウス（左列）と、患者ＩｇＧ投与マウス（右列）の、ＩｇＧ投与から１２時間後の膵臓のＨ＆Ｅ染色（上段）と、Ｇｒ１の染色（下段）の結果を示す。観察像中のスケールバーは上段では５０μｍ、下段では２０μｍを示す。 FIG. 1A shows the results of H & E staining of pancreas 12 hours after IgG administration (upper row) and Gr1 staining (lower row) of control IgG-treated mice (left column) and patient IgG-treated mice (right column). . The scale bar in the observed image shows 50 μm in the upper stage and 20 μm in the lower stage.
　図１Ｂは、対照ＩｇＧ（三角）及び患者ＩｇＧ（丸）の投与量と、浮腫面積との相関関係を示す。図中のカットオフ値（２１．１％）は、対照ＩｇＧ又はＰＢＳ投与マウスでの浮腫面積の平均値に、標準偏差（ＳＤ）の２倍の値を加えた値である。 FIG. 1B shows the correlation between the dose of control IgG (triangle) and patient IgG (circle) and the edema area. The cutoff value (21.1%) in the figure is a value obtained by adding a value twice the standard deviation (SD) to the average value of the edema area in the control IgG or PBS-administered mice.
　図１Ｃは、対照ＩｇＧ投与マウス（左）と、患者ＩｇＧ投与マウス（右）の、ＩｇＧ投与から１２時間後の膵臓の壊死又は出血の病理組織的グレードと、Ｇｒ１陽性細胞数を示す（＊ｐ＜０．０５、＊＊ｐ＜０．００５：ｐａｉｒｅｄ　Ｓｔｕｄｅｎｔ’ｓ　ｔｅｓｔによる）。 FIG. 1C shows the histopathological grade of pancreatic necrosis or hemorrhage 12 hours after IgG administration and the number of Gr1-positive cells in control IgG-treated mice (left) and patient IgG-treated mice (right) ( * p <0.05, ** p <0.005: according to Paired Student's test).
　図１Ｂに示す通り、患者ＩｇＧ投与マウスと、対照ＩｇＧ投与マウスとで、血清中のヒトＩｇＧ濃度に有意な差はなかった（１９．４ｍｇ／ｍｌと２１．４ｍｇ／ｍｌ）。一方で、患者ＩｇＧのマウスへの投与により膵臓が損傷し、図１Ａに示す通り、投与後１２時間で損傷が最大となった。対照ＩｇＧのマウスへの投与では、図１Ａに示す通り、投与後１２時間で膵臓の損傷は生じなかった。 As shown in FIG. 1B, there was no significant difference in serum human IgG concentrations between patient IgG-administered mice and control IgG-administered mice (19.4 mg / ml and 21.4 mg / ml). On the other hand, pancreas was damaged by administration of patient IgG to mice, and the damage was maximized 12 hours after administration as shown in FIG. 1A. Administration of control IgG to mice did not cause pancreatic damage 12 hours after administration, as shown in FIG. 1A.
　組織学的観察では、腺房壊死、出血及びＧｒ１陽性多形核白血球の浸潤が、患者ＩｇＧを投与したマウスの膵臓で観察された（図１Ａ）。 In histological observation, acinar necrosis, hemorrhage, and infiltration of Gr1-positive polymorphonuclear leukocytes were observed in the pancreas of mice administered with patient IgG (FIG. 1A).
　浮腫の面積を定量したところ、患者ＩｇＧ投与マウスでは浮腫形成が認められたが、対照ＩｇＧ投与マウスでは浮腫形成は認められなかった（図１Ｂ）。 When the area of edema was quantified, edema formation was observed in patient IgG-administered mice, but no edema formation was observed in control IgG-administered mice (FIG. 1B).
　膵臓における壊死、出血、及びＧｒ１陽性細胞の浸潤は、患者ＩｇＧ投与マウスにおいて、対照ＩｇＧ投与マウスよりも有意に大きかった（図１Ｃ）。
１．５．結果と考察２（図２Ａ及び図２Ｂ）
　図２Ａ及びＢに結果を示す試験は、患者ＩｇＧの病原性が、膵臓組織への直接的な結合により生じるのか否かを確認する試験である。 Necrosis, hemorrhage, and infiltration of Gr1-positive cells in the pancreas were significantly greater in patient IgG-treated mice than in control IgG-treated mice (FIG. 1C).
1.5. Results and discussion 2 (FIGS. 2A and 2B)
The test whose results are shown in FIGS. 2A and 2B is a test to confirm whether the pathogenicity of patient IgG is caused by direct binding to pancreatic tissue.
　患者ＩｇＧ又は対照ＩｇＧの皮下投与から１２時間後に、マウスの膵臓の組織切片を採取し、免疫組織化学的染色により染色し、観察した。免疫組織化学的染色では、抗ヒトＩｇＧ抗体、抗ヒトＩｇＧ１抗体又は抗ヒトＩｇＧ４抗体を一次抗体として用い、常法により、ヒトＩｇＧ、ヒトＩｇＧ１又はヒトＩｇＧ４を染色した。 Twelve hours after subcutaneous administration of patient IgG or control IgG, mouse pancreas tissue sections were collected, stained by immunohistochemical staining, and observed. In immunohistochemical staining, anti-human IgG antibody, anti-human IgG1 antibody or anti-human IgG4 antibody was used as a primary antibody, and human IgG, human IgG1 or human IgG4 was stained by a conventional method.
　また、前記の、マウスの膵臓の組織切片を免疫蛍光染色により染色し、蛍光顕微鏡により観察した。免疫蛍光染色では、フルオロセインイソシアネートがコンジュゲートされたウサギ抗ヒトＩｇＧ抗体、ウサギ抗アミラーゼ抗体又はウサギ抗コラーゲンＩＶ抗体を抗体として、常法により、ヒトＩｇＧ、アミラーゼ又はコラーゲンＩＶを染色した。 Also, the mouse pancreas tissue section was stained by immunofluorescence staining and observed with a fluorescence microscope. In immunofluorescence staining, human IgG, amylase or collagen IV was stained by a conventional method using a rabbit anti-human IgG antibody, rabbit anti-amylase antibody or rabbit anti-collagen IV antibody conjugated with fluorescein isocyanate.
　図２Ａは、対照ＩｇＧ投与マウス（左列）と、患者ＩｇＧ投与マウス（右列）の、ＩｇＧ投与から１２時間後の膵臓でのＩｇＧ（上段）、ＩｇＧ１（中段）、ＩｇＧ４（下段）の免疫組織化学的染色の結果を示す。観察像中のスケールバーは２０μｍを示す。 FIG. 2A shows immunization of IgG (upper), IgG1 (middle), and IgG4 (lower) in pancreas 12 hours after IgG administration in control IgG-treated mice (left column) and patient IgG-treated mice (right column). The results of histochemical staining are shown. The scale bar in the observed image indicates 20 μm.
　図２Ｂは、対照ＩｇＧ投与マウスと患者ＩｇＧ投与マウスの、膵臓組織の免疫蛍光染色の結果を示す。観察像中のスケールバーは２０μｍを示す。 FIG. 2B shows the results of immunofluorescence staining of pancreatic tissue of control IgG-administered mice and patient IgG-administered mice. The scale bar in the observed image indicates 20 μm.
　図２Ｂの上段の左端は、対照ＩｇＧ投与マウスの膵臓組織での、免疫蛍光染色されたヒトＩｇＧの観察像を示す。ヒトＩｇＧはマウスの膵臓では観察されなかった。 2B shows an observed image of immunofluorescent-stained human IgG in the pancreatic tissue of a control IgG-treated mouse. Human IgG was not observed in the mouse pancreas.
　図２Ｂの上段の左から２つ目は、患者ＩｇＧ投与マウスの膵臓組織での、免疫蛍光染色されたヒトＩｇＧの観察像を示す。図２Ｂの上段の左から３つ目は、患者ＩｇＧ投与マウスの膵臓組織での、図２Ｂの上段の左から２つ目と同じ観察領域における、免疫蛍光染色されたアミラーゼの観察像を示す。図２Ｂの上段の右端は、上記のヒトＩｇＧの観察像と、上記のアミラーゼの観察像とを重ね合わせた像である。 The second from the left in the upper part of FIG. 2B shows an observation image of human IgG stained with immunofluorescence in the pancreatic tissue of a patient IgG-administered mouse. The third from the left in the upper part of FIG. 2B shows an observation image of amylase stained with immunofluorescence in the same observation region as that in the second from the left in the upper part of FIG. The right end of the upper stage of FIG. 2B is an image obtained by superimposing the above observed image of human IgG and the above observed image of amylase.
　図２Ｂの下段の左から１つ目は、患者ＩｇＧ投与マウスの膵臓組織での、免疫蛍光染色されたヒトＩｇＧの観察像を示す。図２Ｂの下段の左から２つ目は、患者ＩｇＧ投与マウスの膵臓組織での、図２Ｂの下段の左から１つ目と同じ観察領域における、免疫蛍光染色されたコラーゲンＩＶの観察像を示す。図２Ｂの下段の右端は、上記のヒトＩｇＧの観察像と、上記のコラーゲンＩＶの観察像とを重ね合わせた像である。 The first from the left in the lower part of FIG. 2B shows an observation image of human IgG stained with immunofluorescence in the pancreatic tissue of a patient IgG-administered mouse. The second from the left in the lower part of FIG. 2B shows an observation image of collagen IV stained with immunofluorescence in the same observation area as the first from the left in the lower part of FIG. . The lower right end of FIG. 2B is an image obtained by superimposing the observed image of the above human IgG and the observed image of the above collagen IV.
　図２Ａに示すように、患者ＩｇＧ投与マウスの全てにおいて、膵臓の腺房の基部及び膵臓組織の小葉間隙においてＩｇＧ及びＩｇＧ４が蓄積していることが観察されたが、対照ＩｇＧ投与マウスの膵臓ではＩｇＧ及びＩｇＧ４は観察されなかった。また、患者ＩｇＧ投与マウスでは１０匹中６匹においてＩｇＧ１が観察されたが、対照ＩｇＧ投与マウスではＩｇＧ１は観察されなかった。 As shown in FIG. 2A, IgG and IgG4 were observed to accumulate in the pancreatic acinar base and pancreatic tissue lobule gap in all patient IgG-treated mice, whereas in control IgG-treated mice pancreas IgG and IgG4 were not observed. In addition, IgG1 was observed in 6 out of 10 mice administered with patient IgG, but IgG1 was not observed in mice administered with control IgG.
　図２Ｂの上段に示す通り、患者ＩｇＧ投与マウスにおいて、ヒトＩｇＧと、膵臓の腺房で発現するアミラーゼとは共存していないことが確認された。また、図２Ｂの下段に示す通り、患者ＩｇＧ投与マウスにおいて、ヒトＩｇＧの大部分と、基底膜又は細胞外マトリクスで発現するコラーゲンＩＶと共存することが確認された。 As shown in the upper part of FIG. 2B, it was confirmed that human IgG and amylase expressed in the pancreatic acinus do not coexist in the patient IgG-administered mouse. In addition, as shown in the lower part of FIG. 2B, it was confirmed that most of the human IgG coexists with collagen IV expressed in the basement membrane or extracellular matrix in the patient IgG-administered mouse.
　以上の結果から、患者ＩｇＧの抗原は、基底膜又は細胞外マトリクスに含まれる成分であると推定される。
＜２．実験２：ＩｇＧ４関連疾患のＩｇＧ１、ＩｇＧ４のマウスへの投与＞
　実験２では、ＩｇＧ４関連疾患患者の血清からＩｇＧのサブクラスＩｇＧ１とＩｇＧ４を精製し、健常マウス新生仔に投与し、投与後の膵臓への影響を観察した。その結果、ＩｇＧ４関連疾患患者の血清から精製したＩｇＧ１とＩｇＧ４はどちらも膵臓に対して病原性を示すこと、ＩｇＧ１の病原性はＩｇＧ４の病原性よりも大きいこと、ＩｇＧ１とＩｇＧ４を併用して投与するとＩｇＧ１の病原性は抑制されることが確認された。
２．１．ヒトＩｇＧの調製
　上記１．１．得たＩｇＧ４関連疾患の患者の血清試料及び対照血清試料から、ＩｇＧ１及びＩｇＧ４を精製した。ＩｇＧ１及びＩｇＧ４の精製は、それぞれ、Ｃａｐｔｕｒｅ　Ｓｅｌｅｃｔ　ＩｇＧ１（Ｈｕ）　ａｆｆｉｎｉｔｙ　ｍａｔｒｉｃｓ（１９１３０３００５，Ｉｎｖｉｔｒｏｇｅｎ製）、及び、Ｃａｐｔｕｒｅ　Ｓｅｌｅｃｔ　ＩｇＧ４（Ｈｕ）　ａｆｆｉｎｉｔｙ　ｍａｔｒｉｃｓ（２９０００５，Ｉｎｖｉｔｒｏｇｅｎ製）を用い、製品の指示書に沿った手順により行った。手順の概要は、ヒト血清試料を前記製品のカラムに負荷し、ＰＢＳで洗浄した。目的とするサブクラス（ＩｇＧ１又はＩｇＧ４）の画分を０．１Ｍグリシン（ｐＨ２．８）により溶出させ、１．５Ｍ　Ｔｒｉｓ（ｐＨ７．５）により中和した。溶出したサブクラス（ＩｇＧ１又はＩｇＧ４）の画分を、ＰＢＳ（ｐＨ７．２）により十分に洗浄しながら、Ａｍｉｃｏｎ　Ｕｌｔｒａ（ＵＦＣ８０５０２４，Ｍｉｌｌｉｐｏｒｅ製）を用いた限外濾過により濃縮した。精製されたＩｇＧサブクラスの濃度を、Ｈｕｍａｎ　ＩｇＧ１　Ｐｌａｔｉｎｕｍ　ＥＬＩＳＡ（ＢＭＳ２０９２，ｅＢｉｏｓｃｉｅｎｃｅ製）、Ｈｕｍａｎ　ＩｇＧ２　Ｐｌａｔｉｎｕｍ　ＥＬＩＳＡ（ＢＭＳ２０９３，ｅＢｉｏｓｃｉｅｎｃｅ製）、Ｈｕｍａｎ　ＩｇＧ３　Ｐｌａｔｉｎｕｍ　ＥＬＩＳＡ（ＢＭＳ２０９４，ｅＢｉｏｓｃｉｅｎｃｅ製）、Ｈｕｍａｎ　ＩｇＧ４　Ｐｌａｔｉｎｕｍ　ＥＬＩＳＡ（ＢＭＳ２０９５，ｅＢｉｏｓｃｉｅｎｃｅ製）を製品の指示書に従って用い、定量した。サブクラスの純度を、上記と同様の手順によりＩｇＡ、ＩｇＭ、ＩｇＥ、他のＩｇＧサブクラス及びタンパク質の混入を求め、確認した。　　　 From the above results, the antigen of patient IgG is presumed to be a component contained in the basement membrane or extracellular matrix.
<2. Experiment 2: IgG1 and IgG4 of IgG4-related disease administration to mice>
In Experiment 2, IgG subclasses IgG1 and IgG4 were purified from sera of patients with IgG4-related diseases and administered to healthy newborn mice, and the effect on the pancreas after administration was observed. As a result, both IgG1 and IgG4 purified from the serum of patients with IgG4-related diseases show pathogenicity to the pancreas, IgG1 pathogenicity is greater than that of IgG4, and administered in combination with IgG1 and IgG4 Then, it was confirmed that the pathogenicity of IgG1 was suppressed.
2.1. Preparation of human IgG Above 1.1. IgG1 and IgG4 were purified from the serum samples of the patients with IgG4-related diseases and the control serum samples obtained. For purification of IgG1 and IgG4, Capture Select IgG1 (Hu) affinity matrix (191303005, manufactured by Invitrogen) and Capture Select IgG4 (Hu) affinity matrix (manufactured by Invitrogen, manufactured by Invitrogen) were used. The procedure was followed. A summary of the procedure was to load a human serum sample onto the product column and wash with PBS. The desired subclass (IgG1 or IgG4) fraction was eluted with 0.1 M glycine (pH 2.8) and neutralized with 1.5 M Tris (pH 7.5). The fraction of the eluted subclass (IgG1 or IgG4) was concentrated by ultrafiltration using Amicon Ultra (UFC805024, manufactured by Millipore) while thoroughly washing with PBS (pH 7.2). The concentration of the purified IgG subclass was determined by using the Human IgG1 Platinum ELISA (BMS2092, eBioscience), Human IgG2 Platinum ELISA (BMS2093, eBioscience), Human IgG3 Platinum ELISA (BMS2094, eBioScientium BMS2094, eBioSc4). Product) was used and quantified according to the product instructions. The purity of the subclass was confirmed by determining the contamination of IgA, IgM, IgE, other IgG subclasses and proteins by the same procedure as described above.
　ＩｇＧ４関連疾患の患者の血清試料から得られたＩｇＧ１、ＩｇＧ４をそれぞれ「患者ＩｇＧ１」、「患者ＩｇＧ４」とした。対照血清試料から得られたＩｇＧ１、ＩｇＧ４をそれぞれ「対照ＩｇＧ１」、「対照ＩｇＧ４」とした。
２．２．動物試験
　マウスとしてＢＡＬＢ／ｃマウスの雄の新生仔を用いた。 IgG1 and IgG4 obtained from serum samples of patients with IgG4-related diseases were designated as “patient IgG1” and “patient IgG4”, respectively. IgG1 and IgG4 obtained from the control serum samples were designated as “control IgG1” and “control IgG4”, respectively.
2.2. Animal test Male newborns of BALB / c mice were used as mice.
　各血清試料から調製されたＩｇＧ１及びＩｇＧ４の一方又は両方、をマウスに皮下投与した。マウスへの投与量は、ＩｇＧ１のみを投与する場合はＩｇＧ１を１０ｍｇ／マウス体重１ｇの量で投与し、ＩｇＧ４のみを投与する場合はＩｇＧ４を１ｍｇ／マウス体重１ｇの量で投与し、ＩｇＧ１とＩｇＧ４とを投与する場合はＩｇＧ１を１０ｍｇ／マウス体重１ｇで投与しＩｇＧ４は１ｍｇ／マウス体重１ｇの量で投与した。
２．３．結果と考察（図３Ａ～図３Ｃ）
　患者ＩｇＧ１及び患者ＩｇＧ４の一方又は両方、或いは、対照ＩｇＧ１及び対照ＩｇＧ４の一方又は両方の皮下投与から１２時間後に、マウスの膵臓の組織切片を採取し、Ｈ＆Ｅ（ヘマトキシリン＆エオシン）染色及び免疫組織化学的染色により染色し、観察した。免疫組織化学的染色では、骨髄細胞分化抗原Ｇｒ１に対する抗体を一次抗体として用い、Ｇｒ１陽性細胞を染色した。免疫組織化学的染色ではまた、ヒトＩｇＧ１又はヒトＩｇＧ４に対する抗体を一次抗体として用い、マウス膵臓組織中のヒトＩｇＧ１又はヒトＩｇＧ４を染色した。 One or both of IgG1 and IgG4 prepared from each serum sample was administered subcutaneously to mice. When administering only IgG1, IgG1 is administered in an amount of 10 mg / mouse body weight of 1 g. When only IgG4 is administered, IgG4 is administered in an amount of 1 mg / mouse body weight of 1 g, and IgG1 and IgG4 Were administered in an amount of 1 mg / mouse weight of 1 g of IgG4.
2.3. Results and discussion (Figures 3A-3C)
Twelve hours after subcutaneous administration of one or both of patient IgG1 and / or patient IgG4, or one or both of control IgG1 and control IgG4, mouse pancreatic tissue sections were collected, H & E (hematoxylin & eosin) staining and immunohistochemistry Stained and observed. In immunohistochemical staining, Gr1-positive cells were stained using an antibody against bone marrow cell differentiation antigen Gr1 as a primary antibody. In the immunohistochemical staining, human IgG1 or human IgG4 in mouse pancreatic tissue was stained using an antibody against human IgG1 or human IgG4 as a primary antibody.
　結果を図３Ａ～図３Ｃに示す。 The results are shown in FIGS. 3A to 3C.
　図３Ａの上段の「対照１」は、１０名の対照のうち特定の１名（対照１）から得た対照ＩｇＧ１及び対照ＩｇＧ４の一方又は両方を投与したマウスの投与１２時間後の膵臓のＨ＆Ｅ染色の観察像である。観察像中のスケールバーは２０μｍを示す。 “Control 1” in the upper part of FIG. 3A shows pancreatic H & E 12 hours after administration of mice administered with one or both of control IgG1 and control IgG4 obtained from a specific one of the 10 controls (control 1). It is an observation image of dyeing | staining. The scale bar in the observed image indicates 20 μm.
　図３Ａの中段及び下段の「患者９」は、１０名のＩｇＧ４関連疾患患者のうち特定の１名（患者９）から得た患者ＩｇＧ１及び患者ＩｇＧ４の一方又は両方を投与したマウスの投与１２時間後の膵臓の、Ｈ＆Ｅ染色の観察像（中段）、及び、Ｇｒ１の免疫組織化学的染色の観察像（下段）である。観察像中のスケールバーは２０μｍを示す。 “Patient 9” in the middle and bottom of FIG. 3A shows 12 hours of administration of a mouse administered with one or both of patient IgG1 and patient IgG4 obtained from one specific patient (patient 9) among 10 IgG4-related disease patients. It is the observation image (lower stage) of H & E staining of the pancreas after, and the observation image (lower stage) of immunohistochemical staining of Gr1. The scale bar in the observed image indicates 20 μm.
　図３Ｂは、対照ＩｇＧ１及び対照ＩｇＧ４の一方又は両方を投与したマウス（左）と、患者ＩｇＧ１及び患者ＩｇＧ４の一方又は両方を投与したマウス（右）の、投与から１２時間後の膵臓の壊死又は出血の病理組織的グレードと、Ｇｒ１陽性細胞数を示す（＊ｐ＜０．０５：ｐａｉｒｅｄ　Ｓｔｕｄｅｎｔ’ｓ　ｔｅｓｔによる）。この結果、患者ＩｇＧ１及び患者ＩｇＧ４はどちらも膵臓に対して病原性を示すこと、患者ＩｇＧ１の病原性は患者ＩｇＧ４の病原性よりも大きいこと、患者ＩｇＧ１と患者ＩｇＧ４を併用して投与すると患者ＩｇＧ１の病原性は抑制されることが確認された。 FIG. 3B shows pancreatic necrosis or 12 hours after administration of mice that received one or both of control IgG1 and control IgG4 (left) and mice that received one or both of patient IgG1 and patient IgG4 (right). The histopathological grade of bleeding and the number of Gr1 positive cells are shown ( * p <0.05: according to Paired Student's test). As a result, both patient IgG1 and patient IgG4 are pathogenic to the pancreas, the pathogenicity of patient IgG1 is greater than the pathogenicity of patient IgG4, and patient IgG1 when administered together with patient IgG1 and patient IgG4 It was confirmed that the pathogenicity of was suppressed.
　図３Ｃは、１０名のＩｇＧ４関連疾患患者のうち特定の１名（患者１）から得た患者ＩｇＧ１及び患者ＩｇＧ４の一方又は両方を投与したマウスの投与１２時間後の膵臓の、ヒトＩｇＧ１又はヒトＩｇＧ４の免疫組織化学的染色の観察像である。観察像中のスケールバーは２０μｍを示す。患者ＩｇＧ１及び患者ＩｇＧ４の両方を投与したマウスでの膵臓のヒトＩｇＧ１染色強度（図３Ｃ上右）は、患者ＩｇＧ１のみを投与したマウスでの膵臓のヒトＩｇＧ１染色強度（図３Ｃ上左）よりも小さいことが確認された。一方、患者ＩｇＧ１及び患者ＩｇＧ４の両方を投与したマウスでの膵臓のヒトＩｇＧ４染色強度（図３Ｃ下右）は、患者ＩｇＧ４のみを投与したマウスでの膵臓のヒトＩｇＧ４染色強度（図３Ｃ上中）と同程度であった。 FIG. 3C shows pancreatic human IgG1 or human 12 hours after administration of mice administered with one or both of patient IgG1 and patient IgG4 from one particular patient (patient 1) out of 10 IgG4-related diseases It is an observation image of immunohistochemical staining of IgG4. The scale bar in the observed image indicates 20 μm. Human IgG1 staining intensity of pancreas in mice administered with both patient IgG1 and patient IgG4 (upper right of FIG. 3C) is greater than human IgG1 staining intensity of pancreas in mice administered only with patient IgG1 (upper left of FIG. 3C) It was confirmed to be small. On the other hand, the human IgG4 staining intensity of the pancreas in the mouse administered with both patient IgG1 and patient IgG4 (lower right in FIG. 3C) is the human IgG4 staining intensity of the pancreas in the mouse administered only with patient IgG4 (upper in FIG. 3C) It was comparable.
　図３Ａ及び図３Ｂからわかるように、患者ＩｇＧ１及び患者ＩｇＧ４の一方又は両方を投与したマウスでは、膵臓において、浮腫形成、腺房壊死、出血及びＧｒ１陽性多形核白血球の浸潤が観察されたのに対して、対照ＩｇＧ１及び対照ＩｇＧ４の一方又は両方を投与したマウスでは膵臓に異常は見られなかった。 As can be seen from FIGS. 3A and 3B, in mice administered with one or both of patient IgG1 and patient IgG4, edema formation, acinar necrosis, hemorrhage and infiltration of Gr1-positive polymorphonuclear leukocytes were observed in the pancreas. In contrast, no abnormality was observed in the pancreas in mice administered with one or both of control IgG1 and control IgG4.
　ＩｇＧ１及びＩｇＧ４はともに病原性を有し、ＩｇＧ１はＩｇＧ４よりも顕著な病原性を有する。しかし、ＩｇＧ１の病原性は、ＩｇＧ４を同時に投与すると顕著に抑制されたことから、ＩｇＧ４はＩｇＧ１の働きを阻害することが示唆される（図３Ｂ）。実際に、ＩｇＧ１のみを投与したマウスでのＩｇＧ１の染色量は、ＩｇＧ１とＩｇＧ４とを併用して投与した場合よりも顕著に多い（図３Ｃ）。一方、ＩｇＧ４のみを投与したマウスでのＩｇＧ４の染色量は、ＩｇＧ１とＩｇＧ４とを併用して投与した場合と同程度であった（図３Ｃ）。このことから、ＩｇＧ１とＩｇＧ４とは膵臓組織へ競合的に結合すること、及び、ＩｇＧ４の膵臓組織への結合親和性は、ＩｇＧ１よりも高いことが分かる。
＜３．実験３：ＡＩＰ患者の膵臓の観察＞
　上記の実験１及び２から、ＩｇＧ４関連疾患の患者のＩｇＧ１及びＩｇＧ４が、膵臓組織に結合して病原性を示すことが判明した。そこで実験３では、自己免疫性膵炎（ＡＩＰ）患者の膵臓において、ＩｇＧ４が蓄積しているかどうかを観察した。観察では、Ｈ＆Ｅ染色によりヒト膵臓組織を染色し、病変がある部分と、病変のない部分を判別した、また、免疫組織化学的染色及び免疫蛍光染色によりヒト膵臓組織中のＩｇＧ４、コラーゲンＩＶ、アミラーゼを可視化した。免疫組織化学的染色及び免疫蛍光染色の手順は実験２と同様である。 Both IgG1 and IgG4 are pathogenic, and IgG1 is more pathogenic than IgG4. However, the pathogenicity of IgG1 was markedly suppressed when IgG4 was administered simultaneously, suggesting that IgG4 inhibits the action of IgG1 (FIG. 3B). In fact, the amount of IgG1 staining in mice administered only with IgG1 is significantly greater than when IgG1 and IgG4 are administered in combination (FIG. 3C). On the other hand, the amount of IgG4 staining in mice administered with IgG4 alone was similar to that obtained when IgG1 and IgG4 were administered in combination (FIG. 3C). From this, it can be seen that IgG1 and IgG4 competitively bind to pancreatic tissue, and that the binding affinity of IgG4 to pancreatic tissue is higher than IgG1.
<3. Experiment 3: Observation of pancreas of AIP patient>
From experiments 1 and 2 above, it was found that IgG1 and IgG4 from patients with IgG4-related disease bind to pancreatic tissue and show pathogenicity. Therefore, in Experiment 3, it was observed whether IgG4 was accumulated in the pancreas of an autoimmune pancreatitis (AIP) patient. In the observation, human pancreatic tissue was stained by H & E staining, and a portion with a lesion was distinguished from a portion without a lesion. Also, IgG4, collagen IV, amylase in human pancreatic tissue were identified by immunohistochemical staining and immunofluorescence staining. Was visualized. The procedure of immunohistochemical staining and immunofluorescent staining is the same as in Experiment 2.
　図４Ａ上段左は、ＡＩＰ病変のある部位（左側）と、病変のない部位（右側）との境界部分から採取した、ＡＩＰ患者の膵臓組織切片をＨ＆Ｅ染色した観察像を示す。図４Ａ上段右は、同様の膵臓組織切片の、ＩｇＧ４に対する免疫組織化学的染色の観察像を示す。スケールバーは２００μｍを示す。 The upper left of FIG. 4A shows an observation image obtained by H & E staining of a pancreatic tissue section of an AIP patient collected from a boundary portion between a site with AIP lesion (left side) and a site without lesion (right side). The upper right of FIG. 4A shows an observation image of immunohistochemical staining for IgG4 of the same pancreatic tissue section. The scale bar indicates 200 μm.
　図４Ａ下段左は、図４Ａ上段右の観察像のうち、病変のある腺房の部分を拡大した観察像である。図４Ａ下段中は、図４Ａ上段右の観察像のうち、病変のある小葉間隙の部分を拡大した観察像である。図４Ａ下段右は、図４Ａ上段右の観察像のうち、病変のない正常な腺房の部分を拡大した観察像である。図４Ａ下段の各観察像においてスケールバーは２０μｍを示す。病変のある膵臓組織では、ＩｇＧ４陽性形質細胞が染色されていることに加えて、腺房の基部及び小葉間隙において、ＩｇＧ４が線状に染色された。一方、病変のない膵臓組織では、このような染色は見られなかった。 The lower left of FIG. 4A is an enlarged image of the lesioned acinar portion in the upper right image of FIG. 4A. The lower part of FIG. 4A is an observation image obtained by enlarging the lesioned leaflet gap portion in the upper right part of FIG. 4A. The lower right side of FIG. 4A is an observation image obtained by enlarging a normal acinar portion without a lesion in the upper right image of FIG. 4A. In each observed image in the lower part of FIG. 4A, the scale bar indicates 20 μm. In the lesioned pancreatic tissue, IgG4 positive plasma cells were stained, and IgG4 was linearly stained at the base of the acinus and the lobule space. On the other hand, such staining was not observed in the pancreatic tissue without lesion.
　図４Ｂの各像は、ＡＩＰ患者の膵臓での病変のある腺房を免疫蛍光染色した観察像を示す。図４Ｂの各像ではスケールバーは２０μｍを示す。 Each image in FIG. 4B shows an observation image obtained by immunofluorescent staining of an acinus having a lesion in the pancreas of an AIP patient. In each image of FIG. 4B, the scale bar indicates 20 μm.
　図４Ｂ上段左は、ＡＩＰ患者の膵臓での病変のある腺房の、免疫蛍光染色されたＩｇＧ４の観察像を示す。図４Ｂ上段中は、図４Ｂ上段左と同じ観察領域の、免疫蛍光染色されたアミラーゼの観察像を示す。図４Ｂ上段右は、図４Ｂ上段左のＩｇＧ４の観察像と、図４Ｂ上段中のアミラーゼの観察像とを重ね合わせた像である。この結果から、ＩｇＧ４はアミラーゼとは共存せず異なる部位に存在することが分かる。 FIG. 4B, upper left, shows an observed image of IgG4 stained with immunofluorescence in the acinus of the AIP patient with a lesion in the pancreas. The upper part of FIG. 4B shows an observation image of amylase stained with immunofluorescence in the same observation area as the upper left part of FIG. 4B. The upper right of FIG. 4B is an image obtained by superimposing the observed image of IgG4 on the upper left of FIG. 4B and the observed image of amylase in the upper part of FIG. 4B. From this result, it can be seen that IgG4 does not coexist with amylase and exists at different sites.
　図４Ｂ下段左は、ＡＩＰ患者の膵臓での病変のある腺房の、免疫蛍光染色されたＩｇＧ４の観察像を示す。図４Ｂ下段中は、図４Ｂ下段左と同じ観察領域の、免疫蛍光染色されたコラーゲンＩＶの観察像を示す。図４Ｂ下段右は、図４Ｂ下段左のＩｇＧ４の観察像と、図４Ｂ下段中のコラーゲンＩＶの観察像とを重ね合わせた像である。図４Ｂ下段右では、赤色のＩｇＧ４と、緑色のコラーゲンＩＶとが重複した部分が、黄色で示される。この結果から、ＩｇＧ４はコラーゲンＩＶと共に腺房の基部に存在することが分かる。 FIG. 4B, lower left, shows an observed image of immunofluorescent-stained IgG4 in an acinus having a lesion in the pancreas of an AIP patient. The lower part of FIG. 4B shows an observation image of collagen IV stained with immunofluorescence in the same observation area as the lower left part of FIG. 4B. The lower right of FIG. 4B is an image obtained by superimposing the observed image of IgG4 on the lower left of FIG. 4B and the observed image of collagen IV in the lower part of FIG. 4B. In the lower right part of FIG. 4B, a portion where red IgG4 and green collagen IV overlap is shown in yellow. From this result, it can be seen that IgG4 is present at the base of the acinar together with collagen IV.
　図４Ｃの各像は、ＡＩＰ患者の膵臓での病変のある小葉間隙を免疫蛍光染色した観察像を示す。図４Ｃの各像ではスケールバーは２０μｍを示す。 Each image in FIG. 4C shows an observation image obtained by immunofluorescent staining of a lobular space with a lesion in the pancreas of an AIP patient. In each image of FIG. 4C, the scale bar indicates 20 μm.
　図４Ｃ左は、ＡＩＰ患者の膵臓での病変のある小葉間隙の、免疫蛍光染色されたＩｇＧ４の観察像を示す。図４Ｃ中は、図４Ｃ左と同じ観察領域の、免疫蛍光染色されたコラーゲンＩＶの観察像を示す。図４Ｃ右は、図４Ｃ左のＩｇＧ４の観察像と、図４Ｃ中のコラーゲンＩＶの観察像とを重ね合わせた像である。図４Ｃ右では、赤色のＩｇＧ４と、緑色のコラーゲンＩＶとが重複した部分が、黄色で示される。この結果から、ＩｇＧ４は、コラーゲンＩＶを発現する細胞外マトリクスに存在することが分かる。 FIG. 4C left shows an observed image of IgG4 stained with immunofluorescence in the lobular space with lesions in the pancreas of an AIP patient. FIG. 4C shows an observation image of collagen IV stained with immunofluorescence in the same observation region as the left of FIG. 4C. The right side of FIG. 4C is an image obtained by superimposing the observed image of IgG4 on the left side of FIG. 4C and the observed image of collagen IV in FIG. 4C. On the right side of FIG. 4C, a portion where red IgG4 and green collagen IV overlap is shown in yellow. From this result, it can be seen that IgG4 exists in the extracellular matrix expressing collagen IV.
　データは示さないが、観察した全てのＡＩＰ患者からの膵臓組織が同様の傾向を示した。 (Data not shown) Pancreatic tissue from all observed AIP patients showed similar trends.
　一方、データは示さないが、健常者の膵臓及び慢性膵炎患者の膵臓ではＩｇＧ４は染色されなかった。
＜４．実験４：ＡＩＰ患者が有する抗体の、抗原の特定＞
　上記実験１～３から、ＡＩＰ患者では、膵臓組織での細胞外マトリクスに特異的に結合するＩｇＧ１及びＩｇＧ４が増加し、それが膵臓の病変を誘導していることが推定された。 On the other hand, data are not shown, but IgG4 was not stained in the pancreas of healthy subjects and the pancreas of patients with chronic pancreatitis.
<4. Experiment 4: Identification of antigen of antibody possessed by AIP patient>
From the above experiments 1 to 3, it was inferred that IgG1 and IgG4 that specifically bind to the extracellular matrix in pancreatic tissue increased in AIP patients, which induced pancreatic lesions.
　そこで本発明者らは、ＡＩＰ患者の血清に含まれる抗体は、膵臓で発現している細胞外マトリクスタンパク質を抗原として認識していると推測した。本発明者らは、抗原を特定するために、文献から既知の、膵臓で発現している細胞外マトリクスタンパク質を、抗原候補として、個別に固相に固定し、ＡＩＰ患者の血清中に、前記細胞外マトリクスタンパク質に結合する抗体が含まれるか否かを、ＥＬＩＳＡ（Ｅｎｚｙｍｅ－Ｌｉｎｋｅｄ　ＩｍｍｕｎｏＳｏｒｂｅｎｔ　Ａｓｓａｙ）法により確認した。ＥＬＩＳＡ法では、ＡＩＰ患者の血清中に含まれ得る、固相に固定された抗原候補と結合するヒトＩｇＧを一次抗体とし、ＨＲＰ（セイヨウワサビペルオキシダーゼ）により標識されたウサギ抗ヒトＩｇＧポリクローナル抗体を二次抗体とした。
４．１．抗原の候補
　抗原候補として、膵臓で発現している細胞外マトリクスタンパク質として既知の以下のタンパク質を試験した。 Therefore, the present inventors speculated that the antibody contained in the serum of the AIP patient recognizes an extracellular matrix protein expressed in the pancreas as an antigen. In order to specify the antigen, the inventors of the present invention individually fixed an extracellular matrix protein expressed in the pancreas known from the literature as an antigen candidate on a solid phase, and in the serum of an AIP patient, Whether or not an antibody that binds to an extracellular matrix protein was contained was confirmed by ELISA (Enzyme-Linked Immunosorbent Assay) method. In the ELISA method, two IgG anti-human IgG polyclonal antibodies labeled with HRP (horseradish peroxidase) are used as primary antibodies, and human IgG that binds to an antigen candidate immobilized on a solid phase, which can be contained in the serum of AIP patients. The secondary antibody was used.
4.1. Antigen candidates As antigen candidates, the following proteins known as extracellular matrix proteins expressed in the pancreas were tested.
　ヒトラミニン－５１１－Ｅ８（８９２０１２，株式会社ニッピ製）（ラミニン－５１１活性断片）
　ヒトコラーゲンＩＶ（ａｂ７５３６，ａｂｃａｍ）
　ヒトフィブロネクチン
　ＧＦＰ（緑色蛍光タンパク質）（ＭＢ－０７５２、ＶＥＣＴＯＲ　ＬＡＢＯＲＡＴＯＲＩＥＳ）
　ヒトＮｏｔｃｈ１（ａｂ６８５８０，ａｂｃａｍ）
　ヒトＡｎｎｅｘｉｎ　Ａ２（ａｂ９３００５，ａｂｃａｍ）
　ヒトＳＤＦ２Ｌ１（ＴＰ３１３１０２，ＯｒｉＧｅｎｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）　ヒトＢｉｌｅ　ｓａｌｔ－ａｃｔｉｖａｔｅｄ　ｌｉｐａｓｅ（ａｂ１６７９６３、ａｂｃａｍ）
４．２．血清試料
　ＡＩＰと診断された９名の患者から血清試料を取得した。 Human laminin-511-E8 (892012, manufactured by Nippi Co., Ltd.) (Laminin-511 active fragment)
Human collagen IV (ab7536, abcam)
Human fibronectin GFP (green fluorescent protein) (MB-0752, VECTOR LABORATORIES)
Human Notch1 (ab68580, abcam)
Human Annexin A2 (ab93005, abcam)
Human SDF2L1 (TP313102, OriGene Technologies) Human Bile salt-activated lipidase (ab167963, abcam)
4.2. Serum samples Serum samples were obtained from 9 patients diagnosed with AIP.
　対照血清試料として、５名の健常者からの血清試料と、５名の疾患患者（３名の膵癌患者、２名の原発性硬化性胆管炎患者）からの血清試料を取得した。
４．３．手順
　以下の試験では、特に明示しない場合は、ＥＬＩＳＡ　Ｓｔａｒｔｅｒ　Ａｃｃｅｓｓｏｒｙ　Ｋｉｔ（Ｅ１０１，Ｂｅｔｈｙｌ　Ｌａｂｏｒａｔｏｒｉｅｓ）を用いた。 As control serum samples, serum samples from 5 healthy subjects and serum samples from 5 disease patients (3 pancreatic cancer patients, 2 primary sclerosing cholangitis patients) were obtained.
4.3. Procedure In the following test, ELISA Starter Access Kit (E101, Bethyl Laboratories) was used unless otherwise specified.
　ＥＬＩＳＡ用コーティングバッファー（ＥＬＩＳＡ　Ｃｏａｔｉｎｇ　Ｂｕｆｆｅｒ）、ＥＬＩＳＡ用洗浄溶液（ＥＬＩＳＡ　Ｗａｓｈ　Ｓｏｌｕｔｉｏｎ）、ＥＬＩＳＡ用ブロッキングバッファー（ＥＬＩＳＡ　Ｂｌｏｃｋｉｎｇ　Ｂｕｆｆｅｒ）、コンジュゲート希釈剤（Ｃｏｎｊｕｇａｔｅ　Ｄｉｌｕｅｎｔ）は全て前記キットの指示書に従い調製した。 The ELISA coating buffer (ELISA Coating Buffer), ELISA wash solution (ELISA Wash Solution), ELISA blocking buffer (ELISA Blocking Buffer), and conjugate diluent (Conjugate Diluent) were all prepared according to the instructions of the kit.
　全ての工程は室温で行った。
４．３．１．抗原候補によるコーティング
　（１）上記の抗原候補の１つを前記キットのＥＬＩＳＡ用コーティングバッファーに希釈して２μｇ／ｍｌ濃度溶液とした。この溶液を、前記キットのマイクロウェルプレートに、１つのウェルあたり１００μｌとなるように加えた。 All steps were performed at room temperature.
4.3.1. Coating with Antigen Candidate (1) One of the above antigen candidates was diluted in the ELISA coating buffer of the kit to obtain a 2 μg / ml concentration solution. This solution was added to the microwell plate of the kit so as to be 100 μl per well.
　（２）上記のマイクロウェルプレートを６０分間インキュベートした。 (2) The above microwell plate was incubated for 60 minutes.
　（３）インキュベート後に各ウェルから溶液を吸引除去した。 (3) After incubation, the solution was removed from each well by suction.
　（４）各ウェルを前記キットのＥＬＩＳＡ用洗浄溶液により洗浄した。具体的には、各ウェルに前記ＥＬＩＳＡ用洗浄溶液を満たし、次いで、前記ＥＬＩＳＡ用洗浄溶液を吸引除去する洗浄操作を３回行った。
４．３．２．ブロッキング
　（１）前記キットのＥＬＩＳＡ用ブロッキングバッファー２００μｌを各ウェルに添加した。 (4) Each well was washed with the ELISA washing solution of the kit. Specifically, each well was filled with the ELISA cleaning solution, and then the cleaning operation for sucking and removing the ELISA cleaning solution was performed three times.
4.3.2. Blocking (1) 200 μl of the ELISA blocking buffer from the kit was added to each well.
　（２）３０分間インキュベートした。 (2) Incubated for 30 minutes.
　（３）インキュベート後、前記ＥＬＩＳＡ用ブロッキングバッファーを除去した後、各ウェルを３回洗浄した。
４．３．３．一次抗体
　（１）前記ＡＩＰ患者血清試料（ｎ＝９）又は前記対照血清試料（ｎ＝１０）を、前記キットのコンジュゲート希釈剤（Ｃｏｎｊｕｇａｔｅ　Ｄｉｌｕｅｎｔ）により１：５０の割合で希釈した。 (3) After incubation, the ELISA blocking buffer was removed, and each well was washed three times.
4.3.3. Primary antibody (1) The AIP patient serum sample (n = 9) or the control serum sample (n = 10) was diluted at 1:50 ratio with the conjugate diluent of the kit (Conjugate Diluent).
　（２）前記希釈により得られた血清（一次抗体）希釈液を、ブロッキング後のマイクロウェルプレートの各ウェルに１００μｌ加えた。 (2) 100 μl of the serum (primary antibody) dilution obtained by the above dilution was added to each well of the microwell plate after blocking.
　（３）６０分間インキュベートした。 (3) Incubated for 60 minutes.
　（４）インキュベート後、前記血清希釈液を除去し、各ウェルを３回洗浄した。
４．３．４．ＨＲＰコンジュゲート二次抗体
　（１）ＨＲＰ（セイヨウワサビペルオキシダーゼ）とコンジュゲートされた二次抗体（ａｂｃａｍ６７５９，ウサギ抗ヒトＩｇＧ　Ｈ＆Ｌ（ＨＲＰ），ポリクローナル）を、前記キットのコンジュゲート希釈剤（Ｃｏｎｊｕｇａｔｅ　Ｄｉｌｕｅｎｔ）により１：２０００の割合で希釈した。 (4) After incubation, the serum dilution was removed and each well was washed three times.
4.3.4. HRP-conjugated secondary antibody (1) Secondary antibody conjugated with HRP (horseradish peroxidase) (abcam6759, rabbit anti-human IgG H & L (HRP), polyclonal) is conjugated diluent of the kit (Conjugate Diluent) Diluted 1: 2000.
　（２）前記希釈により得られた二次抗体希釈液を、血清（一次抗体）を接触させたマイクロウェルプレートの各ウェルに１００μｌ加えた。 (2) 100 μl of the secondary antibody dilution obtained by the above dilution was added to each well of a microwell plate in contact with serum (primary antibody).
　（３）６０分間インキュベートした。 (3) Incubated for 60 minutes.
　（４）インキュベート後、前記二次抗体希釈液を除去し、各ウェルを５回洗浄した。
４．３．５．酵素基質反応
　（１）製造元の推奨条件に従ってＴＭＢ（３，３’，５，５’－テトラメチルベンジジン）溶液を調製した。 (4) After incubation, the secondary antibody dilution was removed, and each well was washed 5 times.
4.3.5. Enzyme substrate reaction (1) A TMB (3,3 ′, 5,5′-tetramethylbenzidine) solution was prepared according to the manufacturer's recommended conditions.
　（２）前記ＴＭＢ溶液を、二次抗体を接触させたマイクロウェルプレートの各ウェルに１００μｌ加えた。 (2) 100 μl of the TMB solution was added to each well of the microwell plate in contact with the secondary antibody.
　（３）５分間～３０分間インキュベートし、ＨＲＰによるＴＭＢの酸化反応を行った。 (3) Incubation was performed for 5 to 30 minutes, and TMB was oxidized with HRP.
　（４）前記酸化反応後に、０．１８Ｍ　Ｈ２ＳＯ４１００μｌを、各ウェルに加えた。 (4) After the oxidation reaction, 100 μl of 0.18 MH 2 SO 4 was added to each well.
　（５）マイクロプレートリーダーを用い、４５０ｎｍの波長にて、前記酸化反応の生成物による呈色を測定した。
４．４．結果と考察
　結果を図５Ａ（抗ヒトラミニン－５１１活性断片抗体）、図５Ｂ（抗ヒトコラーゲンＩＶ抗体）、図５Ｃ（抗ヒトフィブロネクチン抗体）、図５Ｄ（抗ＧＦＰ抗体）に示す。ヒトラミニン－５１１活性断片を抗原とした試験では、試験するＡＩＰ患者血清試料及び対照血清試料の母数を更に増やし、図５Ａに示すように、ＡＩＰ患者血清試料ではｎ＝４０、対照血清試料ではｎ＝４０とした。同様に、ヒトコラーゲンＩＶを抗原とした試験では、試験するＡＩＰ患者血清試料及び対照血清試料の母数を更に増やし、図５Ｂに示すように、ＡＩＰ患者血清試料ではｎ＝４０、対照血清試料ではｎ＝４０とした。 (5) Using a microplate reader, the coloration due to the product of the oxidation reaction was measured at a wavelength of 450 nm.
4.4. Results and Discussion The results are shown in FIG. 5A (anti-human laminin-511 active fragment antibody), FIG. 5B (anti-human collagen IV antibody), FIG. 5C (anti-human fibronectin antibody), and FIG. 5D (anti-GFP antibody). In the test using human laminin-511 active fragment as an antigen, the population of AIP patient serum sample and control serum sample to be tested was further increased. As shown in FIG. 5A, n = 40 for AIP patient serum sample and n for control serum sample. = 40. Similarly, in the test using human collagen IV as an antigen, the population of the AIP patient serum sample and the control serum sample to be tested is further increased, and as shown in FIG. 5B, n = 40 for the AIP patient serum sample and for the control serum sample. n = 40.
　図５Ａ～図５Ｄにおいて縦軸は、各抗原候補に対する抗体の血清中濃度に比例する４５０ｎｍでの吸光度の相対値を示す。カットオフ値は、前記対照血清試料の、各抗原候補に対する抗体の血清中濃度の平均値に、標準偏差（ＳＤ）の２倍の値を加えた値である。抗原候補に対する抗体の血清中濃度が、前記カットオフ値よりも高い場合に、抗原候補に対する抗体に関し陽性であると判断することができる。 5A to 5D, the vertical axis represents the relative absorbance at 450 nm, which is proportional to the serum concentration of the antibody against each antigen candidate. The cut-off value is a value obtained by adding a value twice the standard deviation (SD) to the average value of the serum concentration of the antibody against each antigen candidate in the control serum sample. When the serum concentration of the antibody against the antigen candidate is higher than the cutoff value, it can be determined that the antibody against the antigen candidate is positive.
　図５Ａに示すように、抗ヒトラミニン－５１１抗体に関して、ＡＩＰ患者血清では４０例中２０例で陽性であったのに対し、対照血清では４０例中に陽性例はなかった。このことから、抗ヒトラミニン－５１１抗体の血清中濃度を指標として、ＡＩＰに罹患していることを検出できることが裏付けられた。また、抗ヒトラミニン－５１１抗体が陽性のＡＩＰ患者血清のうち１例では、血中ＩｇＧ濃度及び血中ＩｇＧ４濃度については、対照血清と実質的な差が無く正常値であったことから、抗ヒトラミニン－５１１抗体の血清中濃度を指標としてＡＩＰを判別する方法は、血中ＩｇＧ濃度及び血中ＩｇＧ４濃度を指標とする方法よりも高感度でＡＩＰを判別することができる可能性がある。 As shown in FIG. 5A, anti-human laminin-511 antibody was positive in 20 of 40 cases in AIP patient serum, whereas no positive case was found in 40 cases in control serum. This confirms that AIP can be detected using the serum concentration of anti-human laminin-511 antibody as an index. Further, in one of the AIP patient sera positive for anti-human laminin-511 antibody, the blood IgG concentration and blood IgG4 concentration were normal values with no substantial difference from the control serum. The method of discriminating AIP using the serum concentration of −511 antibody as an index may be able to discriminate AIP with higher sensitivity than the method using blood IgG concentration and blood IgG4 concentration as an index.
　一方、図５Ｂ、５Ｃ及び５Ｄに示す結果から、抗ヒトコラーゲンＩＶ抗体、抗ヒトフィブロネクチン抗体、及び、抗ＧＦＰ抗体に関しては、ＡＩＰ患者血清と対照血清とで血清中濃度に差異は認められず、ＡＩＰの判別指標として有用でないことが判明した。 On the other hand, from the results shown in FIGS. 5B, 5C and 5D, regarding the anti-human collagen IV antibody, the anti-human fibronectin antibody, and the anti-GFP antibody, there is no difference in the serum concentration between the AIP patient serum and the control serum, It was found that it is not useful as a discrimination index for AIP.
　図示しないが、抗ヒトＮｏｔｃｈ１抗体、抗ヒトＡｎｎｅｘｉｎ　Ａ２抗体、抗ヒトＳＤＦ２Ｌ１抗体、抗ヒトＢｉｌｅ　ｓａｌｔ－ａｃｔｉｖａｔｅｄ　ｌｉｐａｓｅ抗体に関しても、ＡＩＰ患者血清と対照血清とで血清中濃度に明確な差異は認められなかった。
＜５．実験５：ラミニン－５１１によるマウスの免疫＞
　上記の実験４の結果は、ヒトラミニン－５１１に対する抗体がＡＩＰの原因物質であると推定される。そこでこの推定を裏付けるべく、実験５では、ヒトラミニン－５１１によりマウスを免疫し、マウスの膵臓での病変の有無を確認した。
５．１．手順
　８週齢の雄マウス（Ｂ６マウス、及び、ＢＡＬＢ／ｃマウス）を麻酔した。ＰＢＳと完全フロイントアジュバント（ＣＦＡ）（２６３８１０，Ｄｉｆｃｏ製）との等量混合液１００μｌ中で乳化させた、１２０μｇのラミニン－５１１－Ｅ８（ヒトラミニン－５１１活性断片）（８９２０１２，株式会社ニッピ製）で、実験を開始した日（第０日）に、麻酔された前記マウスの両方の後足蹠に皮下投与により投与して免疫し、第２８日及び第５６日に、第０日の免疫に用いたのと同じ量の、ＣＦＡ中のラミニン－５１１－Ｅ８によりブーストを行った。 Although not shown, there is no clear difference in serum concentration between AIP patient serum and control serum for anti-human Notch1 antibody, anti-human Annexin A2 antibody, anti-human SDF2L1 antibody, and anti-human Bile salt-activated lipidase antibody. It was.
<5. Experiment 5: Immunization of mice with laminin-511>
From the results of Experiment 4 above, it is presumed that an antibody against human laminin-511 is the causative agent of AIP. In order to support this assumption, in Experiment 5, mice were immunized with human laminin-511 to confirm the presence or absence of lesions in the pancreas of the mice.
5.1. Procedure Eight week old male mice (B6 mice and BALB / c mice) were anesthetized. 120 μg of laminin-511-E8 (human laminin-511 active fragment) (892012, manufactured by Nippi Co., Ltd.) emulsified in 100 μl of an equal volume of PBS and complete Freund's adjuvant (CFA) (263810, manufactured by Difco) On the day when the experiment was started (day 0), both hind footpads of the anesthetized mice were immunized by subcutaneous administration and were used for day 0 and day 56 immunizations. Boosting was done with the same amount of laminin-511-E8 in CFA.
　対照群では、同じ体積の、ＣＦＡにより乳化させた１２０μｇのオボアルブミン（ＯＶＡ）を、ラミニン－５１１－Ｅ８の前記乳化液に代えて用いる以外は同様の手順及びスケジュールでマウスを処理した。 In the control group, mice were treated in the same procedure and schedule except that 120 μg of ovalbumin (OVA) emulsified with CFA was used instead of the emulsion of laminin-511-E8.
　３回目の免疫から２８日後に試験を終了した。試料血清は、最初の投与の前にマウスから採取した血液の血清（免疫前血清）と、屠殺後に採取した血液の血清である。試料血清は分析まで－８０℃に保存した。また、３回目の免疫から２８日後にマウスの膵臓の組織切片を採取し、Ｈ＆Ｅ染色により染色し観察した。 The test was terminated 28 days after the third immunization. Sample sera are blood serum (pre-immune serum) collected from mice prior to the first dose and blood serum collected after sacrifice. Sample serum was stored at −80 ° C. until analysis. Tissue sections of mouse pancreas were collected 28 days after the third immunization and stained with H & E staining for observation.
　上記の操作を、ヒトラミニン－５１１活性断片を投与した試験群、及び、対照群のそれぞれについてｎ＝３となるよう実施した。
５．２．結果と考察
　Ｈ＆Ｅ染色により染色した膵臓の組織切片の観察像を図６に示す。図６左が対照群の観察像、図６右がヒトラミニン－５１１活性断片（ｉ５１１ラミニン）により免疫した試験群の観察像である。ヒトラミニン－５１１活性断片により免疫した試験群のマウスでは膵臓に細胞浸潤の病変が形成されたのに対して、対照群のマウスでは膵臓に異常は見られなかった。ヒトラミニン－５１１活性断片により免疫した試験群のマウスでも、膵臓以外の部位には異常は見られなかった。図６に示す観察像を得たマウス個体以外のマウス個体においても、同様の傾向であった。
＜６．実験６：ＩｇＧ４関連腎疾患の患者が有する抗ラミニン抗体の分析＞
　実験４に記載のＡＩＰ患者からの血清試料又は対照血清試料を用い、抗原候補としてヒトラミニン－５２１を用いて、実験４に記載の手順により抗ヒトラミニン－５２１抗体の血清中濃度を求めた。 The above operation was performed so that n = 3 for each of the test group to which the human laminin-511 active fragment was administered and the control group.
5.2. Results and Discussion FIG. 6 shows an observation image of a tissue section of the pancreas stained by H & E staining. FIG. 6 left is an observation image of a control group, and FIG. 6 right is an observation image of a test group immunized with a human laminin-511 active fragment (i511 laminin). In the test group of mice immunized with human laminin-511 active fragment, cell infiltrated lesions were formed in the pancreas, whereas in the control group of mice, no abnormality was found in the pancreas. Even in the test group of mice immunized with the human laminin-511 active fragment, no abnormalities were observed in the regions other than the pancreas. The same tendency was observed in mouse individuals other than the mouse individual that obtained the observation image shown in FIG.
<6. Experiment 6: Analysis of anti-laminin antibodies possessed by patients with IgG4-related kidney disease>
Using the serum sample from the AIP patient described in Experiment 4 or a control serum sample and using human laminin-521 as an antigen candidate, the serum concentration of the anti-human laminin-521 antibody was determined by the procedure described in Experiment 4.
　ヒトラミニン－５２１としてＨｕｍａｎ　ｒｅｃｏｍｂｉｎａｎｔ　ｌａｍｉｎｉｎ　５２１（ＢＬＡ－ＬＮ５２１－０２、Ｂｉｏ　Ｌａｍｉｎａ）を用いた。 Human recombinant laminin 521 (BLA-LN521-02, Bio Lamina) was used as human laminin-521.
　結果を図７に示す。 Results are shown in FIG.
　９例のＡＩＰ患者のうち、ＩｇＧ４関連腎疾患を持つのは１例のみであった。この１例のＩｇＧ４関連腎疾患を持つＡＩＰ患者からの血清試料のみが、抗ヒトラミニン－５２１抗体を有意に高濃度で含んでいた。 Of the 9 AIP patients, only 1 had IgG4-related kidney disease. Only the serum sample from this one AIP patient with IgG4-related kidney disease contained a significantly higher concentration of anti-human laminin-521 antibody.
　この結果から、ＩｇＧ４関連腎疾患に関しては、抗ヒトラミニン－５２１抗体の血清中濃度が判別の指標として有効である可能性が示唆された。
＜７．実験７：ＩｇＧ４関連腎疾患患者ＩｇＧのマウスへの投与＞
　実験６において抗ヒトラミニン－５２１抗体陽性であることが確認されたＩｇＧ４関連腎疾患患者の血清から、実験１と同様の手順でＩｇＧを調製した。このＩｇＧを「ＩｇＧ４関連腎疾患患者ＩｇＧ」とした。 From these results, it was suggested that the serum concentration of anti-human laminin-521 antibody may be effective as an indicator of discrimination for IgG4-related renal diseases.
<7. Experiment 7: Administration of IgG4 Related Kidney Disease Patient IgG to Mice>
IgG was prepared from the serum of an IgG4-related renal disease patient who was confirmed to be positive for anti-human laminin-521 antibody in Experiment 6 by the same procedure as in Experiment 1. This IgG was designated as “IgG4-related kidney disease patient IgG”.
　一方、実験１で調製した対照ＩｇＧを用意した。 Meanwhile, a control IgG prepared in Experiment 1 was prepared.
　また、腎疾患を有していないＩｇＧ４関連疾患患者の血清から、実験１と同様の手順でＩｇＧを調製した。このＩｇＧを「腎疾患のないＩｇＧ４関連疾患患者ＩｇＧ」とした。 In addition, IgG was prepared from the serum of an IgG4-related disease patient who has no kidney disease by the same procedure as in Experiment 1. This IgG was designated as “IgG4 related disease patient IgG without renal disease”.
　上記のＩｇＧ４関連腎疾患患者ＩｇＧ、対照ＩｇＧ、及び、腎疾患のないＩｇＧ４関連疾患患者ＩｇＧを、それぞれ、実験１と同様の手順でＢＡＬＢ／ｃマウスの雄の新生仔に皮下投与した。 The above IgG4-related kidney disease patient IgG, control IgG, and IgG4-related disease patient IgG without kidney disease were each subcutaneously administered to male newborns of BALB / c mice in the same procedure as in Experiment 1.
　投与から１２時間後に、実験１と同様の手順で、マウスの腎臓を採取し免疫組織化学的染色によりヒトＩｇＧ４を染色し観察した。 12 hours after administration, mouse kidneys were collected in the same manner as in Experiment 1, and human IgG4 was stained and observed by immunohistochemical staining.
　結果を図８に示す。 The results are shown in FIG.
　図８左側にＩｇＧ４関連腎疾患患者ＩｇＧを皮下投与したマウスの腎臓の観察像を示し、図８右側に対照ＩｇＧを皮下投与したマウスの腎臓のヒトＩｇＧ４染色観察像を示す。上段は、腎臓全体のヒトＩｇＧ４染色観察像を示し、下段は糸状体のヒトＩｇＧ４染色観察像を示す。図示しないが、腎疾患のないＩｇＧ４関連疾患患者ＩｇＧを皮下投与した場合の腎臓の観察像は、対照ＩｇＧと同様であった。 FIG. 8 shows the observation image of the kidney of the mouse subcutaneously administered with IgG4 related kidney disease patient IgG on the left side of FIG. 8, and shows the human IgG4 staining observation image of the kidney of the mouse subcutaneously administered with the control IgG on the right side of FIG. The upper row shows the human IgG4 staining observation image of the whole kidney, and the lower row shows the human IgG4 staining observation image of the filamentous body. Although not shown, the observed image of the kidney when IgG4 related disease patient IgG without renal disease was subcutaneously administered was the same as that of the control IgG.
　図８の結果から、ＩｇＧ４関連腎疾患患者ＩｇＧをマウスに投与した場合、腎臓の糸状体の間質にヒトＩｇＧ４が結合するのに対して、対照ＩｇＧをマウスに投与した場合にはそのような状態は生じないことが分かる。 From the results in FIG. 8, when IgG4 related kidney disease patient IgG is administered to mice, human IgG4 binds to the interstitium of kidney filaments, whereas when control IgG is administered to mice, such It can be seen that no condition occurs.
　一方、ラミニンβ２鎖の遺伝子異常によりＰｉｅｒｓｏｎ症候群が生じることが知られている。Ｐｉｅｒｓｏｎ症候群は、腎不全（メザンギウム増殖性糸状球腎炎）、神経障害（筋力低下）、縮瞳を含む。上記のＩｇＧ４関連腎疾患患者ＩｇＧを取得した、抗ヒトラミニン－５２１抗体陽性のＩｇＧ４関連腎疾患患者は、腎生検からメザンギウム増殖性糸状球腎炎であることが確認されており、筋力低下も確認されていることから、Ｐｉｅｒｓｏｎ症候群と症状、病理が合致する。従って、このＩｇＧ４関連腎疾患患者は、ラミニンβ２鎖に対する自己抗体を有しており、それが原因でＩｇＧ４関連腎疾患の症状が現れていると推測される。
＜８．実験８：ＡＩＰ患者の血清中の抗ラミニン５１１－Ｅ８自己抗体の測定＞
８．１．試料
　ＩｇＧ４－ＲＤ　２０１１の診断基準又はＡＩＰの診断基準を満たす５１名の治療中のＡＩＰ患者からの血清試料を取得した。２５名の健常者及び８６名の疾患患者（４９名の、組織学的に確認された癌患者、及び、３９名の、癌以外の疾患患者）から、対照の血清試料を取得した。ＡＩＰ患者５１名のうち１０名からの血清試料と、対照者１１２名のうち１０名からの血清試料をそれぞれトレーニング群として用い、残りをバリデーション群として用いた。全ての血清試料は－８０℃にて保存した。
８．２．ＥＬＩＳＡの手順
　抗原の候補物質であるヒトラミニン５１１－Ｅ８として、ヒト由来リコンビナントラミニン－５１１－Ｅ８（８９２０１２，株式会社ニッピ製）を用いた。ラミニン５１１－Ｅ８は、ラミニンのα鎖、β鎖及びγ鎖のＣ末端領域がヘテロ三量体を形成した活性フラグメントであり、インテグリンへの結合能力を有する。ヒトラミニン－５１１－Ｅ８に対する血清抗体を、ＥＬＩＳＡ　Ｓｔａｒｔｅｒ　Ａｃｃｅｓｓｏｒｙ　Ｋｉｔ（Ｅ１０１，Ｂｅｔｈｙｌ　Ｌａｂｏｒａｔｏｒｉｅｓ）を該キットの指示の通り用いるＥＬＩＳＡ（Ｅｎｚｙｍｅ－Ｌｉｎｋｅｄ　ＩｍｍｕｎｏＳｏｒｂｅｎｔ　Ａｓｓａｙ）により定量した。定量方法の概要は以下の通りである。 On the other hand, it is known that Pierson syndrome is caused by a genetic abnormality of laminin β2 chain. Pierson's syndrome includes renal failure (mesangial proliferating filamentous nephritis), neuropathy (muscular weakness), and miosis. Anti-human laminin-521 antibody-positive IgG4-related kidney disease patients who have obtained the above IgG4-related kidney disease patient IgG have been confirmed to have mesangial proliferative fibronephritis by renal biopsy, and muscle weakness has also been confirmed. Therefore, Pierson's syndrome matches symptoms and pathology. Therefore, this IgG4-related renal disease patient has an autoantibody against laminin β2 chain, which is presumed to cause symptoms of IgG4-related renal disease.
<8. Experiment 8: Measurement of anti-laminin 511-E8 autoantibodies in serum of AIP patients>
8.1. Samples Serum samples from 51 in-treatment AIP patients who met the diagnostic criteria for IgG4-RD 2011 or the diagnostic criteria for AIP were obtained. Control serum samples were obtained from 25 healthy individuals and 86 disease patients (49 histologically confirmed cancer patients and 39 non-cancer disease patients). Serum samples from 10 of 51 AIP patients and serum samples from 10 of 112 controls were used as training groups, and the rest were used as validation groups. All serum samples were stored at -80 ° C.
8.2. ELISA Procedure Human-derived recombinant traminin-511-E8 (892012, manufactured by Nippi Co., Ltd.) was used as human laminin 511-E8, which is an antigen candidate substance. Laminin 511-E8 is an active fragment in which the C-terminal regions of laminin α chain, β chain, and γ chain form a heterotrimer, and has the ability to bind integrin. Serum antibodies to human laminin-511-E8 were quantified by ELISA (Enzyme-Linked Immunosorbent Assay) using an ELISA Starter Accessory Kit (E101, Ethyl Laboratories) as directed by the kit. The outline of the quantification method is as follows.
　ヒトラミニン－５１１－Ｅ８を前記キットのＥＬＩＳＡ用コーティングバッファーに希釈して２μｇ／ｍｌ濃度溶液とし、この溶液を、前記キットのマイクロタイタープレートに、１つのウェルあたり１００μｌとなるように加え、室温で１時間インキュベートしてコーティングした。０．０５％　Ｔｗｅｅｎ２０含有Ｔｒｉｓ緩衝生理食塩水（洗浄液）で前記プレートを５回洗浄した後、１％ウシ血清アルブミン含有Ｔｒｉｓ緩衝生理食塩水で前記プレートをコーティングし、１００μＬの、１：２０希釈血清で室温にて３０分間インキュベートした。前記洗浄液で５回洗浄した後、前記プレートを、１００μＬの、ＨＲＰ（セイヨウワサビペルオキシダーゼ）とコンジュゲートされたヤギ抗ヒトＩｇＧ抗体（１：４０００；ａｂ６７５９，Ａｂｃａｍ）と、室温にて１時間インキュベートした。前記洗浄液で３回洗浄した後、前記プレートをＴＭＢ（３，３’，５，５’－テトラメチルベンジジン）により３分間インキュベートし、４５０ｎｍでの吸光度を測定して結合物量を定量した。ヒトラミニン－５１１－Ｅ８でコーティングしていない対照ウェルを、各血清試料についてのＥＬＩＳＡにおけるネガティブコントロールとして用いた。各アッセイはｎ＝３で行い、ヒトラミニン－５１１－Ｅ８でコーティングしたウェルでの吸光度の平均値から、対照ウェルでの吸光度の平均値を引いた値を、血清抗体のヒトラミニン－５１１－Ｅ８への特異的結合量、すなわち、血清中の抗ヒトラミニン－５１１－Ｅ８　ＩｇＧ抗体レベルとした。
８．３．結果
　トレーニング群である１０名のＡＩＰ患者、及び、１０名の対照（膵臓癌患者３名、原発性硬化性胆管炎患者２名、健常者５名）からの血清試料の、抗ヒトラミニン－５１１－Ｅ８　ＩｇＧ抗体レベルの測定結果を図９Ａに示す。カットオフ値（光学密度単位［ＯＤ］）は、前記対照からの血清試料での抗ヒトラミニン－５１１－Ｅ８　ＩｇＧ抗体レベルの平均値に、標準偏差（ＳＤ）の３倍の値を加えた値である。１０名のＡＩＰ患者のうち６名からの血清試料が、ヒトラミニン－５１１－Ｅ８に対するＩｇＧ抗体が陽性であったのに対して、１０名の対照者の全員がヒトラミニン－５１１－Ｅ８に対するＩｇＧ抗体が陰性であった。 Human laminin-511-E8 was diluted in the ELISA coating buffer of the kit to obtain a 2 μg / ml concentration solution, and this solution was added to the microtiter plate of the kit so as to be 100 μl per well. Incubate for time to coat. The plate was washed five times with 0.05% Tween 20-containing Tris buffered saline (washing solution), then coated with 1% bovine serum albumin-containing Tris buffered saline, and 100 μL of 1:20 diluted serum. And incubated at room temperature for 30 minutes. After washing 5 times with the washing solution, the plate was incubated with 100 μL of goat anti-human IgG antibody conjugated with HRP (horseradish peroxidase) (1: 4000; ab6759, Abcam) for 1 hour at room temperature. . After washing with the washing solution three times, the plate was incubated with TMB (3,3 ′, 5,5′-tetramethylbenzidine) for 3 minutes, and the absorbance at 450 nm was measured to quantify the amount of bound substance. Control wells not coated with human laminin-511-E8 were used as negative controls in the ELISA for each serum sample. Each assay was performed at n = 3, and the average value of absorbance in wells coated with human laminin-511-E8 minus the average value of absorbance in control wells was used to determine the serum antibody to human laminin-511-E8. Specific binding amount, ie, the level of anti-human laminin-511-E8 IgG antibody in serum.
8.3. Results Anti-human laminin-511 of serum samples from 10 AIP patients in training group and 10 controls (3 patients with pancreatic cancer, 2 patients with primary sclerosing cholangitis, 5 healthy people). The measurement result of the E8 IgG antibody level is shown in FIG. 9A. The cut-off value (optical density unit [OD]) is a value obtained by adding three times the standard deviation (SD) to the mean value of anti-human laminin-511-E8 IgG antibody level in the serum sample from the control. is there. Serum samples from 6 of 10 AIP patients were positive for IgG antibodies to human laminin-511-E8, whereas all 10 controls had IgG antibodies to human laminin-511-E8. Negative.
　バリデーション群の４１名のＡＩＰ患者、及び、１０２名の対照（健常者２０名、ＡＩＰ以外の疾患患者８２名）からの血清試料の、抗ヒトラミニン－５１１－Ｅ８　ＩｇＧ抗体レベルの測定結果を図９Ｂに示す。カットオフ値（光学密度単位［ＯＤ］）は、前記対照からの血清試料での抗ヒトラミニン－５１１－Ｅ８　ＩｇＧ抗体レベルの平均値に、標準偏差（ＳＤ）の３倍の値を加えた値である。４１名のＡＩＰ患者のうち２０名からの血清試料が、抗ヒトラミニン－５１１－Ｅ８　ＩｇＧ抗体を有していた（陽性であった）のに対して、１０２名の対照者のうち２名のみが、抗ヒトラミニン－５１１－Ｅ８　ＩｇＧ抗体陽性であった。 FIG. 9B shows the measurement results of anti-human laminin-511-E8 IgG antibody levels of serum samples from 41 AIP patients in the validation group and 102 controls (20 healthy subjects, 82 disease patients other than AIP). Shown in The cut-off value (optical density unit [OD]) is a value obtained by adding 3 times the standard deviation (SD) to the mean value of anti-human laminin-511-E8 IgG antibody level in the serum sample from the control. is there. Serum samples from 20 of 41 AIP patients had anti-human laminin-511-E8 IgG antibody (positive), whereas only 2 out of 102 controls Anti-human laminin-511-E8 IgG antibody was positive.
　上記のトレーニング群及びバリデーション群の測定結果をまとめると、ヒトラミニン－５１１－Ｅ８に対するＩｇＧ抗体陽性は、ＡＩＰ患者血清では２６／５１（５１．０％）であったのに対して、対照血清では２／１１２（１．８％）であった（Ｐ＜０．００１）。 Summarizing the measurement results of the training group and the validation group, the IgG antibody positive against human laminin-511-E8 was 26/51 (51.0%) in the serum of AIP patients, whereas it was 2 in the control serum. / 112 (1.8%) (P <0.001).
　５１名のＡＩＰ患者の血清試料の測定結果、及び、被験者の臨床像を下記の表４にまとめた。 The measurement results of the serum samples of 51 AIP patients and the clinical image of the subjects are summarized in Table 4 below.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　抗ヒトラミニン－５１１－Ｅ８抗体陽性のＡＩＰ患者では、陰性のＡＩＰ患者と比較して、悪性腫瘍及びアレルギー疾患の頻度が有意に低いことが判明した。 It was found that the frequency of malignant tumors and allergic diseases was significantly lower in anti-human laminin-511-E8 antibody positive AIP patients than in negative AIP patients.
　抗ヒトラミニン－５１１－Ｅ８抗体陽性のＡＩＰ患者２６名の全員が悪性腫瘍を合併していなかったのに対して、陰性のＡＩＰ患者２５名中８名が悪性腫瘍を合併していた。 All 26 of the AIP patients positive for anti-human laminin-511-E8 antibody did not have a malignant tumor, whereas 8 of 25 negative AIP patients had a malignant tumor.
　抗ヒトラミニン－５１１－Ｅ８抗体陽性のＡＩＰ患者２６名中３名のみがアレルギー疾患を合併していたのに対して、陰性のＡＩＰ患者２５名中１２名がアレルギー疾患を合併していた。 Only 3 out of 26 AIP patients positive for anti-human laminin-511-E8 antibody had allergic disease, whereas 12 out of 25 negative AIP patients had allergic disease.
　また、造影ＣＴによる膵画像の比較から、抗ヒトラミニン－５１１－Ｅ８抗体陽性のＡＩＰ患者では、膵臓の頭部病変を示す患者の割合が、陰性のＡＩＰ患者と比較して有意に低いことが判明した。
＜９．実験９：抗ラミニン５１１－Ｅ８自己抗体陰性のＡＩＰ患者における抗インテグリン自己抗体の測定＞
　膵臓においてラミニン５１１は、インテグリンα６β１又はインテグリンα３β１と結合することが知られている。本発明者らは、ＡＩＰ患者では、インテグリンα６β１又はインテグリンα３β１も自己抗原となるという仮説を立てた。
９．１．試料
　実験８と同じ、５１名のＡＩＰ患者及び１１２名の対照からの血清試料を用いた。
９．２．ＥＬＩＳＡ
　抗原候補物質としてインテグリンα６β１又はインテグリンα３β１を用いた以外は、実験８と同じ手順で、ＥＬＩＳＡにより、血清試料中の抗インテグリンα６β１抗体及び抗インテグリンα３β１抗体を定量した。 In addition, comparison of pancreatic images by contrast-enhanced CT revealed that the proportion of patients with pancreatic head lesions was significantly lower in anti-human laminin-511-E8 antibody-positive AIP patients than in negative AIP patients did.
<9. Experiment 9: Measurement of anti-integrin autoantibodies in AIP patients negative for anti-laminin 511-E8 autoantibodies>
Laminin 511 is known to bind to integrin α6β1 or integrin α3β1 in the pancreas. The inventors hypothesized that in AIP patients, integrin α6β1 or integrin α3β1 is also a self-antigen.
9.1. Samples Serum samples from 51 AIP patients and 112 controls as in Experiment 8 were used.
9.2. ELISA
The anti-integrin α6β1 antibody and the anti-integrin α3β1 antibody in the serum sample were quantified by ELISA in the same procedure as in Experiment 8, except that integrin α6β1 or integrin α3β1 was used as an antigen candidate substance.
　インテグリンα６β１として、リコンビナントヒトインテグリンα６β１（Ｒ＆ＤＳｙｓｔｅｍｓ、米国ミネソタ州、製品番号７８０９－Ａ６）を用いた。 As the integrin α6β1, recombinant human integrin α6β1 (R & D Systems, Minnesota, USA, product number 7809-A6) was used.
　インテグリンα３β１として、リコンビナントヒトインテグリンα３β１（Ｒ＆ＤＳｙｓｔｅｍｓ、米国ミネソタ州、製品番号２８４０－Ａ３）を用いた。
９．３．結果
　２６名の抗ラミニン５１１－Ｅ８抗体陽性ＡＩＰ患者、２５名の抗ラミニン５１１－Ｅ８抗体陰性ＡＩＰ患者、及び、１１２名の対照からの血清試料での、抗インテグリンα６β１　ＩｇＧ抗体レベルの測定結果を図１０に、抗インテグリンα３β１　ＩｇＧ抗体レベルの測定結果を図１１に、それぞれ示す。カットオフ値（光学密度単位［ＯＤ］）は、前記対照からの血清試料での抗インテグリンα６β１　ＩｇＧ抗体レベル又は抗インテグリンα３β１　ＩｇＧ抗体レベルの平均値に、標準偏差（ＳＤ）の３倍の値を加えた値である。 As integrin α3β1, recombinant human integrin α3β1 (R & D Systems, Minnesota, USA, product number 2840-A3) was used.
9.3. Results Measurements of anti-integrin α6β1 IgG antibody levels in serum samples from 26 anti-laminin 511-E8 antibody positive AIP patients, 25 anti-laminin 511-E8 antibody negative AIP patients, and 112 controls. FIG. 10 shows the measurement results of the anti-integrin α3β1 IgG antibody level, respectively. The cut-off value (optical density unit [OD]) is the mean value of anti-integrin α6β1 IgG antibody level or anti-integrin α3β1 IgG antibody level in the serum sample from the control, which is 3 times the standard deviation (SD). It is the added value.
　図１０に示す通り、２６名の抗ラミニン５１１－Ｅ８抗体陽性ＡＩＰ患者、及び、１１２名の対照は、全員が、抗インテグリンα６β１　ＩｇＧ抗体陰性であった。２５名の抗ラミニン５１１－Ｅ８抗体陰性ＡＩＰ患者のうち４名が、抗インテグリンα６β１　ＩｇＧ抗体陽性であった。 As shown in FIG. 10, 26 anti-laminin 511-E8 antibody positive AIP patients and 112 controls were all negative for anti-integrin α6β1 IgG antibody. Four of 25 anti-laminin 511-E8 antibody negative AIP patients were positive for anti-integrin α6β1 IgG antibody.
　図１１に示す通り、５１名のＡＩＰ患者及び１１２名の対照は、全て、抗インテグリンα３β１　ＩｇＧ抗体陰性であった。 As shown in FIG. 11, all 51 AIP patients and 112 controls were negative for anti-integrin α3β1 IgG antibody.
　以上の結果は、ラミニン５１１に加えてインテグリンα６β１が、一部のＡＩＰ患者において自己抗原であることを示す。このことは、ラミニン５１１とインテグリンα６β１との結合が、ＡＩＰでの自己抗体の標的であることを示唆する。 The above results indicate that integrin α6β1 is an autoantigen in some AIP patients in addition to laminin 511. This suggests that the binding of laminin 511 and integrin α6β1 is a target for autoantibodies at AIP.
　５１名のＡＩＰ患者を、２６名の抗ラミニン５１１－Ｅ８抗体陽性ＡＩＰ患者（抗インテグリンα６β１抗体は陰性）と、４名の抗インテグリンα６β１抗体陽性ＡＩＰ患者（抗ラミニン５１１－Ｅ８抗体は陰性）と、２１名の抗ラミニン５１１－Ｅ８抗体陰性抗インテグリンα６β１抗体陰性ＡＩＰ患者に分類し、各群のＡＩＰ患者の臨床像を下記の表５にまとめた。なお、抗ラミニン５１１－Ｅ８抗体と抗インテグリンα６β１抗体が両方とも陽性の被験者はいなかった。 51 AIP patients were divided into 26 anti-laminin 511-E8 antibody positive AIP patients (anti-integrin α6β1 antibody negative) and 4 anti-integrin α6β1 antibody positive AIP patients (anti-laminin 511-E8 antibody negative). The 21 anti-laminin 511-E8 antibody negative anti-integrin α6β1 antibody negative AIP patients were classified, and the clinical features of each group of AIP patients are summarized in Table 5 below. There were no subjects who were positive for both anti-laminin 511-E8 antibody and anti-integrin α6β1 antibody.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　２６名の抗ラミニン５１１－Ｅ８抗体陽性ＡＩＰ患者は、悪性腫瘍を合併している者が皆無であった。一方、４名の抗インテグリンα６β１抗体陰性ＡＩＰ患者のうち２名（５０％）が悪性腫瘍を合併していた。また、２１名の抗ラミニン５１１－Ｅ８抗体陰性抗インテグリンα６β１抗体陰性ＡＩＰ患者のうち６名（２９％）が悪性腫瘍を合併していた。このことから、ＡＩＰ患者において、血清中の抗ラミニン５１１－Ｅ８抗体レベル及び抗インテグリンα６β１抗体レベルを指標として、悪性腫瘍の発症の可能性を予測することが可能である。例えば、抗インテグリンα６β１抗体陽性のＡＩＰ患者は、悪性腫瘍を合併する可能性が高いため、全身をスクリーニングする必要性が高いと言える。
＜１０．実験１０：膵画像の比較＞
　図１２上段は、抗ラミニン５１１－Ｅ８抗体陽性ＡＩＰ患者の造影ＣＴによる膵画像である。図１２下段は、抗インテグリンα６β１抗体陽性ＡＩＰ患者の造影ＣＴによる膵画像である。各画像中の矢印は病変箇所を指す。 None of the 26 anti-laminin 511-E8 antibody positive AIP patients had malignant tumors. On the other hand, of 4 anti-integrin α6β1 antibody negative AIP patients, 2 (50%) had malignant tumors. Of 21 anti-laminin 511-E8 antibody negative anti-integrin α6β1 antibody negative AIP patients, 6 (29%) had malignant tumors. From this, in AIP patients, it is possible to predict the possibility of developing malignant tumors using the anti-laminin 511-E8 antibody level and anti-integrin α6β1 antibody level in serum as indices. For example, an AIP patient who is positive for anti-integrin α6β1 antibody is highly likely to have a malignant tumor, so it can be said that it is highly necessary to screen the whole body.
<10. Experiment 10: Comparison of pancreatic images>
The upper part of FIG. 12 is a pancreatic image by contrast-enhanced CT of an anti-laminin 511-E8 antibody positive AIP patient. The lower part of FIG. 12 is a pancreatic image obtained by contrast CT of an anti-integrin α6β1 antibody-positive AIP patient. The arrow in each image indicates the lesion site.
　図１２上段に示すように、抗ラミニン５１１－Ｅ８抗体陽性ＡＩＰ患者の膵画像では、膵臓の体部及び尾部に病変が認められた。図１２下段に示すように、抗インテグリンα６β１抗体陽性ＡＩＰ患者の膵画像では、膵臓の体部に病変が認められ、これは膵癌患者の膵画像と類似しており、抗インテグリンα６β１抗体陰性ＡＩＰ患者４７例では同様の膵画像は皆無であった。
＜１１．実験１１：抗ラミニン５１１－Ｅ８抗体力価の治療による低減＞
１１．１．手順
　実験８で抗ラミニン５１１－Ｅ８抗体陽性であった２６名のＡＩＰ患者のうち５名について、ステロイドを用いたＡＩＰ治療の前後での、血清中の抗ラミニン５１１－Ｅ８抗体レベルを調べた。抗体レベルの測定は実験８と同様の方法で行った。 As shown in the upper part of FIG. 12, in the pancreatic image of an anti-laminin 511-E8 antibody positive AIP patient, lesions were observed in the body part and tail part of the pancreas. As shown in the lower part of FIG. 12, in the pancreatic image of an anti-integrin α6β1 antibody-positive AIP patient, a lesion was observed in the body part of the pancreas, which is similar to the pancreatic image of a pancreatic cancer patient, and an anti-integrin α6β1 antibody-negative AIP patient There were no similar pancreatic images in 47 cases.
<11. Experiment 11: Reduction of anti-laminin 511-E8 antibody titer by treatment>
11.1. Procedure Five of 26 AIP patients who were positive for anti-laminin 511-E8 antibody in Experiment 8 were tested for serum anti-laminin 511-E8 antibody levels before and after AIP treatment with steroids. The antibody level was measured in the same manner as in Experiment 8.
　各患者はＡＩＰと診断された後、ステロイドの１種であるプレドニゾンの経口投与による治療を６～８カ月間にわたり受けた。
１１．２．結果
　５名の抗ラミニン５１１－Ｅ８抗体陽性ＡＩＰ患者の、ステロイド治療前後での、血清中の抗ラミニン５１１－Ｅ８抗体レベルを図１３に示す。ステロイド治療により各ＡＩＰ患者の血清中の抗ラミニン５１１－Ｅ８抗体力価はカットオフ値未満に低減された。また、図示しないが、各ＡＩＰ患者の血清中の抗ラミニン５１１－Ｅ８抗体力価の低減に伴い膵画像所見も改善した。このことは、血清中の抗ラミニン５１１－Ｅ８抗体は、ＡＩＰ治療の効果の指標としても有用であることを裏付ける。 Each patient was treated with oral administration of prednisone, a steroid, for 6-8 months after being diagnosed with AIP.
11.2. Results FIG. 13 shows the serum anti-laminin 511-E8 antibody levels of five anti-laminin 511-E8 antibody positive AIP patients before and after steroid treatment. Steroid treatment reduced the anti-laminin 511-E8 antibody titer in the serum of each AIP patient below the cut-off value. Although not shown, pancreatic imaging findings also improved with the reduction of the anti-laminin 511-E8 antibody titer in the serum of each AIP patient. This confirms that anti-laminin 511-E8 antibody in serum is also useful as an indicator of the effect of AIP treatment.
　本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。 All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims (15)

  1. 　ＩｇＧ４関連疾患の検査方法であって、
    　ＩｇＧ４関連疾患の指標として、検体中の、ラミニンと免疫学的に反応する抗体を検出する抗ラミニン抗体検出工程、及び／又は
    　ＩｇＧ４関連疾患の指標として、検体中の、インテグリンと免疫学的に反応する抗体を検出する抗インテグリン抗体検出工程
    を含む方法。     A test method for IgG4-related diseases,
    Anti-laminin antibody detection step for detecting an antibody that immunologically reacts with laminin as an indicator of IgG4-related disease, and / or immunological reaction with integrin in a sample as an indicator of IgG4-related disease A method comprising an anti-integrin antibody detection step of detecting an antibody to be detected.
  2. 　前記ＩｇＧ４関連疾患が、自己免疫性膵炎であり、
    　抗ラミニン抗体検出工程における前記抗体が、ラミニン－５１１と免疫学的に反応する抗体である、請求項１に記載の方法。     The IgG4-related disease is autoimmune pancreatitis;
    The method according to claim 1, wherein the antibody in the anti-laminin antibody detection step is an antibody that immunologically reacts with laminin-511.
  3. 　前記ＩｇＧ４関連疾患が、自己免疫性膵炎であり、
    　抗インテグリン抗体検出工程における前記抗体が、インテグリンα６β１と免疫学的に反応する抗体である、請求項１又は２に記載の方法。     The IgG4-related disease is autoimmune pancreatitis;
    The method according to claim 1 or 2, wherein the antibody in the anti-integrin antibody detection step is an antibody that immunologically reacts with integrin α6β1.
  4. 　前記ＩｇＧ４関連疾患が、ＩｇＧ４関連腎疾患であり、
    　抗ラミニン抗体検出工程における前記抗体が、ラミニン－５２１と免疫学的に反応する抗体である、請求項１に記載の方法。     The IgG4-related disease is IgG4-related kidney disease;
    The method according to claim 1, wherein the antibody in the anti-laminin antibody detection step is an antibody that immunologically reacts with laminin-521.
  5. 　前記抗ラミニン抗体検出工程が、ラミニンを抗原として用いて前記抗体を検出することを含む、請求項１～４のいずれか１項に記載の方法。 The method according to any one of claims 1 to 4, wherein the anti-laminin antibody detection step comprises detecting the antibody using laminin as an antigen.
  6. 　自己免疫性膵炎の検査方法であって、
    　自己免疫性膵炎の指標として、検体の血液試料中の、ラミニン－５１１と免疫学的に反応する抗体を検出する抗ラミニン抗体検出工程、及び
    　自己免疫性膵炎の指標として、検体の血液試料中の、インテグリンα６β１と免疫学的に反応する抗体を検出する抗インテグリン抗体検出工程
    　を含む方法。     A test method for autoimmune pancreatitis,
    As an index of autoimmune pancreatitis, an anti-laminin antibody detection step for detecting an antibody immunologically reactive with laminin-511 in a sample blood sample, and as an index of autoimmune pancreatitis in a sample blood sample And an anti-integrin antibody detection step of detecting an antibody that immunologically reacts with integrin α6β1.
  7. 　ラミニンを含む、ＩｇＧ４関連疾患を検査するための検査試薬。 Test reagents for testing IgG4-related diseases, including laminin.
  8. 　前記ＩｇＧ４関連疾患が、自己免疫性膵炎であり、
    　前記ラミニンが、ラミニン－５１１である、請求項７に記載の検査試薬。     The IgG4-related disease is autoimmune pancreatitis;
    The test reagent according to claim 7, wherein the laminin is laminin-511.
  9. 　前記ＩｇＧ４関連疾患が、ＩｇＧ４関連腎疾患であり、
    　前記ラミニンが、ラミニン－５２１である、請求項７に記載の検査試薬。     The IgG4-related disease is IgG4-related kidney disease;
    The test reagent according to claim 7, wherein the laminin is laminin-521.
  10. 　前記ラミニンを、固相に固定された形態で含む、請求項７～９のいずれか１項に記載の検査試薬。 The test reagent according to any one of claims 7 to 9, comprising the laminin in a form fixed to a solid phase.
  11. 　インテグリンを含む、ＩｇＧ４関連疾患を検査するための検査試薬。 Test reagents for testing IgG4-related diseases, including integrins.
  12. 　ラミニン－５１１とインテグリンα６β１とを固相に固定された形態で含む、自己免疫性膵炎の検査試薬。 A test reagent for autoimmune pancreatitis comprising laminin-511 and integrin α6β1 fixed in a solid phase.
  13. 　ＩｇＧ４関連疾患に対する治療の効果を評価する方法であって、
    　ＩｇＧ４関連疾患に対する治療が施された被検動物から得た検体中の、ラミニンと免疫学的に反応する抗体を検出する抗ラミニン抗体検出工程、及び／又は、
    　ＩｇＧ４関連疾患に対する治療が施された被検動物から得た検体中の、インテグリンと免疫学的に反応する抗体を検出する抗インテグリン抗体検出工程
    を含む方法。     A method for assessing the effect of treatment on an IgG4-related disease comprising:
    An anti-laminin antibody detection step of detecting an antibody that immunologically reacts with laminin in a specimen obtained from a test animal treated for an IgG4-related disease; and / or
    A method comprising an anti-integrin antibody detection step of detecting an antibody that immunologically reacts with an integrin in a specimen obtained from a test animal treated for an IgG4-related disease.
  14. 　ＩｇＧ４関連疾患の治療薬の候補物質をスクリーニングする方法であって、
    　試験物質を作用させた動物から得た検体中の、ラミニンと免疫学的に反応する抗体を検出する抗ラミニン抗体検出工程と、
    　前記試験物質を作用させたことにより前記検体中の前記抗体が減少した場合に、前記試験物質を、ＩｇＧ４関連疾患の治療薬の候補物質として選抜する選抜工程と
    を含む方法。     A method for screening candidate substances for therapeutic agents for IgG4-related diseases, comprising:
    An anti-laminin antibody detection step for detecting an antibody that immunologically reacts with laminin in a specimen obtained from an animal on which a test substance has been allowed to act;
    And a selection step of selecting the test substance as a candidate substance for a therapeutic drug for IgG4-related disease when the antibody in the specimen decreases due to the action of the test substance.
  15. 　ＩｇＧ４関連疾患の非ヒトモデル動物を作製する方法であって、
    　非ヒト動物に、ラミニンと免疫学的に反応する抗体を投与する抗ラミニン抗体投与工程、及び
    　非ヒト動物を、ラミニンを抗原として免疫するラミニン免疫工程
    のうち少なくとも一方を含む方法。     A method for producing a non-human animal model of an IgG4-related disease comprising:
    A method comprising at least one of an anti-laminin antibody administration step of administering an antibody that immunologically reacts with laminin to a non-human animal, and a laminin immunization step of immunizing a non-human animal with laminin as an antigen.
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