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WO2004094638A1 - Vasculitis antigen and method of diagnosing vasculitis - Google Patents

Vasculitis antigen and method of diagnosing vasculitis Download PDF

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Publication number
WO2004094638A1
WO2004094638A1 PCT/JP2004/005729 JP2004005729W WO2004094638A1 WO 2004094638 A1 WO2004094638 A1 WO 2004094638A1 JP 2004005729 W JP2004005729 W JP 2004005729W WO 2004094638 A1 WO2004094638 A1 WO 2004094638A1
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WO
WIPO (PCT)
Prior art keywords
antibody
vasculitis
subject
peptide
antigen peptide
Prior art date
Application number
PCT/JP2004/005729
Other languages
French (fr)
Japanese (ja)
Inventor
Tomohiro Kato
Kusuki Nishioka
Shoichi Ozaki
Rie Karasawa
Original Assignee
St. Marianna University, School Of Medicine
Medical And Biological Laboratories Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by St. Marianna University, School Of Medicine, Medical And Biological Laboratories Co., Ltd. filed Critical St. Marianna University, School Of Medicine
Priority to JP2005505770A priority Critical patent/JPWO2004094638A1/en
Publication of WO2004094638A1 publication Critical patent/WO2004094638A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the invention of this application relates to an antigen peptide for human vasculitis, a polynucleotide encoding the peptide, and a method for diagnosing vasculitis using the same.
  • a group of diseases mainly composed of vascular inflammation such as polyarteritis nodosa (so-called vasculitis syndrome) is generally intractable and has a poor prognosis, and elucidation of the etiology is a social demand.
  • vasculitis Diseases that are included in vasculitis include polygenetic nodular arthritis, Zegener's granulomatosis, microscopic polyarteritis nodosa, allergic granulomatous vasculitis, polyarteritis nodosa, temporal arteritis, Takayasu's arteritis, rheumatoid arthritis, Baja disease, anti-phospholipid antibody syndrome, Kawasaki disease, Henoch-Schenlein purpura, Libedo purpura, crescentic nephritis, pulmonary capillary vasculitis, chiag- Many diseases are known, such as Strauss syndrome.
  • vasculitis can be associated with collagen diseases such as dermatomyositis, mixed connective tissue disease, scleroderma, Behcet's disease, Siegren's syndrome, and systemic lupus erythematosus.
  • collagen diseases such as dermatomyositis, mixed connective tissue disease, scleroderma, Behcet's disease, Siegren's syndrome, and systemic lupus erythematosus.
  • An autoimmune mechanism is thought to be involved in the pathogenesis, but the details are unknown.
  • the invention of this application has been made in view of the circumstances described above, and has an object to provide a novel antigen peptide which is effective for diagnosing vasculitis.
  • This application provides the following inventions (1) to (17) to solve the above problems.
  • vasculitis antigen peptide according to the invention (1) which is a human peroxyredoxin 2 homolog having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide according to the invention (3) which has the nucleotide sequence of SEQ ID NO: 1 and encodes the human peroxyredoxin 2 homologue according to the invention (2).
  • An antibody that binds to the antigenic peptide of the invention (1) is detected in a biological sample collected from a subject, and a subject in which the antibody is present in a biological sample in a greater amount than a healthy subject is identified as a vasculitis patient or A method for diagnosing human vasculitis, characterized by determining that the patient has a high risk of vasculitis.
  • An antigen peptide that binds to the antibody of the invention (6) is detected in a biological sample collected from a subject, and the subject whose antigen peptide is present in the biological sample in a greater amount than in a healthy subject is subjected to vasculitis.
  • a method for diagnosing human vasculitis which is determined to be a patient or a person at high risk for vasculitis.
  • a diagnostic kit for vasculitis comprising:
  • a diagnostic kit for vasculitis comprising:
  • a diagnostic kit for vasculitis comprising at least the antibody of the invention (6) and / or the labeled antibody of the invention (7).
  • a diagnostic kit for vasculitis comprising:
  • a diagnostic kit for vasculitis comprising:
  • a therapeutic agent for vasculitis comprising human peroxyredoxin or a fragment containing an antigenic determinant thereof, which is immobilized on a carrier insoluble in blood.
  • a method for treating or preventing vasculitis comprising the following steps.
  • step (b) returning the blood or a fraction thereof to the patient after step (a).
  • the inventors of the present application analyzed vascular endothelial cells by comprehensive analysis of autoantigen peptides specific to vascular endothelial cells by two-dimensional electrophoresis (for example, Elec trophores is 22: 3019-3025, 2001). Identified the vasculitis antigen peptide to which the sera of patients with inflammation reacted, which was identified as peroxyredoxin or The present invention was completed by finding that it was thioredoxin peroxidase.
  • Peroxyredoxin or thioredoxin peroxidase is known as an enzyme protein related to its antioxidant activity and cellular response to oxygen stress, etc., but it has a relationship with vasculitis, especially in the plasma of patients with vasculitis. It has never been known that it reacts specifically with the antibody in it.
  • the invention of this application is based on the novel antigenic peptide described above, an antibody against the peptide, and a polynucleotide encoding the same.
  • protein and “peptide” mean a molecule composed of a plurality of amino acid residues linked to each other by an amide bond (peptide bond).
  • Polynucleotide is a nucleoside phosphoric acid ester (ATP, GTP, CTP, UTP; or dATP, dGTP, dCTP, dUTP) containing at least 100 N-daricoside-linked pyridine or pyrimidine.
  • Oligonucleotide refers to a linked molecule of 2-99.
  • an antibody recognizing peroxyredoxin means an antibody that binds to the antigen peptide of the invention (1), but does not include an antibody that binds nonspecifically to peroxyredoxin.
  • the “antibody” of the inventions (6) and (7) means a polyclonal antibody or a monoclonal antibody prepared using the antigen peptide of the invention (1) as an immunogen.
  • the vasculitis antigen peptide of the invention (1) is Peroxired oxin (PRDX).
  • PRDX Peroxired oxin
  • This PRDX may also be referred to as Thioredoxin peroxidase.
  • PRDX includes: PRDX1 (GenBank / NM_002574), PRDX2 (GenBank / NM—005809), PRDX 3 (GenBank / ⁇ —006793), PRDX4 (GenBank / Martian—006406), PRDX5 (GenBank / NM—0112094), PRDX6 (GenBank / NM—004905) Force S Known.
  • GenBank / BC003609, GenBank / BC007 107, etc. are known as red-red redoxin peroxidase.
  • the vasculitis antigen of the present invention (1) includes all of these known PRDXs, but a PRDX2 homolog (GenBank / BC000452) having the amino acid sequence of SEQ ID NO: 2 is particularly preferable (the invention (2)).
  • antigenic peptides can be expressed, for example, by preparing RNA from the recombinant expression vector having the polynucleotide of the invention (3) by in vitro transcription and performing in vitro translation using this as a type III to express the peptide in vitro. it can. E. coli, Bacillus subtilis, etc.
  • eukaryotic cells such as prokaryotic cells
  • eukaryotic cells such as yeast cells, insect cells, and mammalian cells to produce transformed cells
  • peptides can be expressed from the transformed cells.
  • the polynucleotide When expressing the antigen peptide by in vitro translation, the polynucleotide is inserted into a vector having an RNA polymerase promoter to prepare a recombinant expression vector, and this vector is used as a rabbit containing the RNA polymerase corresponding to the promoter.
  • the antigen peptide When added to an in vitro translation system such as a reticulocyte lysate or a wheat germ extract, the antigen peptide can be produced in vitro.
  • the RNA polymerase promoter include T7, T3, and SP6.
  • vectors containing these RNA polymerase promoters include pKAl, pCDM8, pT3 / T718, ⁇ 7 / 319, pBluescript II and the like.
  • expression is performed by recombining polynucleotides into a vector having an origin, a promoter, a ribosome binding site, a DNA cloning site, a terminator, etc. that can be replicated in microorganisms. After preparing a vector, transforming a host cell with this expression vector, and culturing the resulting transformant, the antigen peptide encoded by the polynucleotide can be expressed from the microorganism. . At this time, it can be expressed as a fusion protein with another protein.
  • expression vectors for Escherichia coli include a pUC system, pBluescript II, a pET expression system, a pGEX expression system, and a pMAL expression system.
  • the polynucleotide When the antigen peptide is expressed in eukaryotic cells, the polynucleotide is inserted into an expression vector for eukaryotic cells having a promoter, a splicing region, a poly (A) addition site, etc. to prepare a recombinant vector.
  • antigenic peptides When introduced into nuclear cells, antigenic peptides can be expressed in transformed eukaryotic cells. it can.
  • Expression vectors, pKAl, pCDM8, pSVK3, pMSG , P SVL, BK - CMV, pBK RSV, EBV vector, pRS, pcDNA3, pMSG, pYES2 such force S can be exemplified.
  • pIND / V5-His, pFLAG-CMV-2, pEGFP-Nl, pEGFP-CI, etc. are used as expression vectors, various tags such as His tag, FLAG tag, myc tag, HA tag, and GFP can be added.
  • An antigen peptide can also be expressed as a fused protein.
  • mammalian cultured cells such as monkey kidney cells C0S7 and Chinese hamster ovary cells CH0, budding yeast, fission yeast, silkworm cells, African egg cells and the like are generally used. Any eukaryotic cell can be used as long as it can express the antigenic peptide of the present invention.
  • known methods such as an electroporation method, a calcium phosphate method, a ribosome method, and a DEAE dextran method can be used.
  • the target peptide After expressing the antigen peptide in prokaryotic cells or eukaryotic cells, the target peptide can be isolated and purified from the culture by a combination of known separation procedures. For example, treatment with denaturing agents such as urea or surfactants, ultrasonic treatment, enzyme digestion, salting-out / solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, Examples include ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.
  • denaturing agents such as urea or surfactants
  • ultrasonic treatment enzyme digestion
  • salting-out / solvent precipitation dialysis
  • centrifugation ultrafiltration
  • gel filtration gel filtration
  • SDS-PAGE isoelectric focusing
  • Examples include ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.
  • the recombinant antigen peptide obtained by the above method also includes a fusion protein with any other protein.
  • a fusion protein with glutathione-1S-transferase (GST) and green fluorescent protein (GFP) can be exemplified.
  • GST glutathione-1S-transferase
  • GFP green fluorescent protein
  • peptides expressed in transformed cells may undergo various modifications in the cells after translation. Therefore, the modified peptide is also included in the scope of the antigen peptide of the first invention.
  • post-translational modifications include N-terminal methionine elimination, acetylation, and sugar chains. Examples thereof include addition, limited degradation by intracellular protease, myristoylation, isoprenylation, and phosphorylation.
  • the antigen peptide obtained by the above method is used as a material for the method of diagnosing vasculitis provided by the present invention.
  • Invention (3) is a polynucleotide (DNA fragment, RNA fragment) encoding the antigenic peptide of invention (1).
  • genomic DNA encoding each peptide (protein), mRNA transcribed from genomic DNA, and cDNA synthesized from mRNA. It may be double-stranded or single-stranded.
  • the sense strand and antisense strand of these genomic DNA, mRNA and cDNA are also included.
  • genomic DNA it also includes its expression control region (promoter, enhancer, sublesser region).
  • CDNA can be obtained by a method of isolating each cDNA using a probe DNA prepared based on a known base sequence.
  • the obtained cDNA is subjected to, for example, PCR (Polymerase Chain Reaction), NASBA (Nucleic acid sequence base amplification), TMA (Transcription-mediated
  • This method can be extended by a commonly used gene amplification method such as the SDA (Strand Displacement Amplification) method.
  • the required amount of each cDNA can be obtained by RT-PCR using the primer set provided by the present invention as a type II mRNA isolated from human cells.
  • a preferred embodiment of the polynucleotide thus obtained is PRDX 2 Polynucleotide (cDNA) having the nucleotide sequence of SEQ ID NO: 1 encoding a homolog (invention (4)).
  • the polynucleotide of the present invention (3) also includes an oligonucleotide consisting of a partially continuous sequence thereof.
  • an oligonucleotide can also be obtained, for example, by cleaving the above-mentioned polynucleotide (cDNA) with an appropriate restriction enzyme.
  • cDNA polynucleotide
  • oligonucleotide includes those labeled with a labeling substance.
  • Labeling can be performed by a radioisotope (RI) method or a non-RI method, but it is preferable to use a non-RI method.
  • RI radioisotope
  • non-RI method examples include a fluorescent labeling method, a biotin labeling method, and a chemiluminescent method, and it is preferable to use a fluorescent labeling method.
  • the fluorescent substance it can be used to select those that can bind with a base portion of the oligonucleotides appropriate, Shiani emissions dyes (e.g., Cy Dye TM series of Cy3, Cy5, etc.), Rodami down 6G reagent, N -Acetoxy-N 2 -acetylaminofluorene (AAF), AAIF (iodine derivative of AAF) and the like can be used.
  • Shiani emissions dyes e.g., Cy Dye TM series of Cy3, Cy5, etc.
  • Rodami down 6G reagent e.g., N -Acetoxy-N 2 -acetylaminofluorene (AAF), AAIF (iodine derivative of AAF) and the like can be used.
  • Invention (5) is a primer set for PCR amplification of the polynucleotide of invention (3), which is designed based on a known nucleotide sequence, and It can be prepared through each step of synthesis and purification.
  • the following points can be pointed out as points to keep in mind when designing primers.
  • the size (number of bases) of the primer is 15-40 bases, preferably 15-30 bases, in view of satisfying specific annealing with type I DNA. However, when performing LA (long accurate) PCR, at least 30 bases are effective. Avoid the complementary sequences between the primers so that one or two pairs of primers consisting of the sense strand (5, terminal) and the antisense strand (3, terminal) do not anneal to each other.
  • the GC content should be about 50%, so that GC-rich or AT-rich is not unevenly distributed in the primer. Since the annealing temperature depends on the melting temperature (Tm), in order to obtain a highly specific PCR product, primers having a Tm value of 55-65 ° C and close to each other are selected. It is also necessary to take care to adjust the final concentration of the primer used in PCR to be about 0.1 to about 1 ⁇ ⁇ .
  • commercially available software for primer design for example, Oligo TM [manufactured by National Bioscience Inc. (USA)], GENETYX [manufactured by Software Development Co., Ltd. (Japan)] and the like can also be used.
  • the antibody of the invention (6) is a polyclonal antibody or a monoclonal antibody, and the whole molecule capable of binding to the epitope of the antigenic peptide of the invention (1), Fab, F (ab ') 2 , Fv fragment and the like are all included. included.
  • the antibody of the invention (7) is an antibody that binds to a different epitope from the antibody of the invention (6).
  • a polyclonal antibody such an antibody can be obtained from serum after immunizing an animal using the antigen peptide or a fragment thereof as an immunogen.
  • serum is collected after introducing the above eukaryotic cell expression vector into the muscle or skin of an animal by injection or gene gun. Can be produced by As animals, mice, rats, egrets, goats, and chickens are used.
  • Monoclonal antibodies can be prepared using known monoclonal antibody preparation methods (“Monoclonal Antibodies”, written by Kamei Nagamune and Hiroshi Terada, Hirokawa Shoten, 1990; “Monoclonal Antibody James W. Goding, third edition, Academic Press, 1996).
  • the antibody of the invention (7) is an antibody that binds to a different epitope from the antibody of the invention (6).
  • Such an antibody is produced as a polyclonal antibody or a monoclonal antibody as described above by using, as an immunogen, a fragment different from the antigen peptide fragment for producing the antibody of the invention (6).
  • the antibodies of the inventions (6) and (7) each include an antibody labeled with a labeling substance.
  • a labeling substance an enzyme, a radioisotope or a fluorescent dye can be used. There are no particular restrictions on the enzyme as long as it satisfies conditions such as a large turnover number, stability even when bound to the antibody, and specific coloring of the substrate.
  • peroxidase peroxidase, monogalactosidase, alkaline phosphatase, gnorecosoxidase, acetylcholinesterase, glucose-16-phosphoryl dehydrogenase, malate dehydrogenase, luciferase and the like can be used.
  • an enzyme inhibitor, a capture enzyme, or the like, or label with ataridinium'ester can be used.
  • the binding between the enzyme and the antibody can be performed by a known method using a crosslinking agent such as a maleimide compound.
  • a known substance can be used depending on the type of the enzyme to be used.
  • peroxidase when peroxidase is used as the enzyme, 3,3 ', 5,5'-tetramethylbenzicine can be used.
  • alkaline phosphatase When alkaline phosphatase is used as the enzyme, paranitrophenol can be used.
  • radioisotopes Those used in normal RIA such as 125 I and 3 H can be used.
  • fluorescent dye those used in a usual fluorescent antibody method, such as fluorescein isothiocyanate (FITC) ⁇ tetramethylrhodamine isothiocyanate (TRITC), can be used.
  • FITC fluorescein isothiocyanate
  • TRITC tetramethylrhodamine isothiocyanate
  • the method for diagnosing human vasculitis according to the invention (8) comprises detecting an antibody that binds to the antigenic peptide of the invention (1) in a biological sample collected from a subject, and detecting the antibody in the biological sample from a healthy subject.
  • Subjects with high levels of vasculitis are determined to be vasculitis patients or high-risk vasculitis patients. That is, since the antigenic peptide of the invention (1) is a peptide that binds to an antibody in a biological sample of a vasculitis patient, the antigenic peptide is reacted with a biological sample of a subject, and more antibodies bind to this antigenic peptide than in a healthy subject.
  • Detection of the antibody can be carried out as various known immunoassays such as RIA, ELISA, EIA, CLIA, CLEIA, and luciferase atsey.
  • the detection in the present invention may be either qualitative or quantitative.
  • the antibody can be used for the purpose of diagnosing and screening vasculitis and also for monitoring the treatment of vasculitis.
  • blood, serum, plasma, blood cells eg, mononuclear cells
  • urine bone marrow fluid, saliva, and the like can be used.
  • other known vasculitis markers can also be used in combination.
  • the serum of the subject is brought into contact with the antigen peptide, and the antigen peptide is reacted with the IgG antibody in the serum of the subject in a liquid phase.
  • a signal of the labeled anti-human IgG antibody may be detected by reacting with a labeled anti-human IgG antibody that specifically binds to the IgG antibody in serum.
  • an enzyme, a radioisotope, a fluorescent dye or the like as exemplified in the above-mentioned labeled antibody can be used.
  • an enzyme add a substrate that decomposes and develops color by the action of the enzyme.
  • the enzyme activity is determined by optically measuring the amount of degradation of the enzyme, and this is converted into the amount of bound antibody.
  • a radioisotope measure the radiation dose emitted by the radioisotope using a scintillation counter or the like.
  • the amount of fluorescence may be measured by a measuring device combined with a fluorescence microscope.
  • the chemical luminescence method it is sufficient to measure the amount of acridinium.ester luminescence.
  • Western plot analysis can be employed.
  • the conjugate of the antigen peptide + the antibody in the biological sample + the labeled anti-human IgG antibody is separated by a known separation method (chromatography, solid phase method, etc.), and the labeled anti-human IgG antibody is separated. Signal may be detected.
  • the diagnostic kit of the invention (13) is provided to enable such a diagnostic method to be performed simply and widely.
  • the diagnostic method of the invention (8) may be carried out as a method (invention (9)) in which one or more antigen peptides are immobilized on a carrier, and the binding of the subject biological sample to the carrier is tested on the carrier. You can also. By immobilizing the antigen peptide on the carrier, unbound labeled binding molecules can be easily removed.
  • the carrier beads, container inner walls, fine particles, porous carriers, magnetic particles, or the like are used. These solid phases utilize materials such as polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, latex, gelatin, agarose, glass, metal, or ceramic. Can be used.
  • Solid phase materials in which a functional group for chemically binding an antibody or the like is introduced to the surface of these solid phase materials are also known.
  • the solid phase and the antibody (or antigen) well-known binding methods such as chemical binding such as poly-L-lysine-glutalaldehyde treatment and physical adsorption can be applied.
  • a diagnostic method is simple and broad.
  • the diagnostic kit of the invention (14) is provided to enable a wide range of implementation.
  • the expression of various antibodies can be analyzed in a short time using about 0.01 ml of the serum of the subject.
  • the diagnostic method according to the invention (10) of the present application comprises detecting an antigen peptide that binds to the antibody of the invention (6) or a labeled antibody thereof in a biological sample of a subject, and the antigen peptide is detected in the sample.
  • Subjects present more than healthy subjects are determined to be vasculitis patients or their high-risk subjects. That is, since the antibody or labeled antibody used here is an antibody that specifically binds to an antigen peptide expressed in vascular endothelial cells exhibiting vasculitis, the antigen peptide that binds to this antibody is used.
  • Biological samples can include blood, serum, plasma, blood cells (eg, mononuclear cells), urine, bone marrow fluid, saliva, and the like.
  • One embodiment of the diagnostic method of the present invention is a method in which the antibody is bound to the antigen peptide in a liquid phase system.
  • the labeled antibody of the invention (6) is brought into contact with a biological sample to bind the labeled antibody and the antigen peptide, the conjugate is separated by the same method as in the invention (8), and the labeling signal is similarly determined.
  • the diagnostic kit according to the invention (15) is provided as a method that enables such a diagnostic method to be carried out simply and widely.
  • Another method of diagnosis in a liquid phase system is the method in which the antibody (primary antibody) of the invention (6) is brought into contact with a biological sample to bind the primary antibody and the antigen peptide, and the invention is labeled with this conjugate.
  • the antibody (secondary antibody) of (7) is allowed to bind, and the labeling signal of the conjugate is detected.
  • an unlabeled secondary antibody may first be bound to the antibody + antigen peptide conjugate, and a labeling substance may be bound to this secondary antibody. This Such binding of the labeling substance to the secondary antibody can be performed, for example, by biotinylation of the secondary antibody and avidinization of the labeling substance.
  • the tertiary antibody so as to bind to the second antibody, primary antibody
  • a monoclonal antibody can be used, or one of the primary antibody and the secondary antibody can be a polyclonal antibody. Separation of the conjugate from the liquid phase and detection of the signal can be performed in the same manner as in the invention (8).
  • the diagnostic kit of the invention (16) is provided as a method which enables simple and wide-ranging implementation of such a diagnostic method.
  • Another embodiment of the diagnostic method of the invention is a method of testing the binding between an antibody and an antigen peptide in a solid phase system.
  • This method using a solid phase system is a preferred method for detecting a trace amount of antigen peptide and simplifying the operation. That is, this solid-phase method involves immobilizing the antibody of the invention (6) on a carrier such as a resin plate or a bead-membrane, binding the antigen peptide to the immobilized antibody, washing away the unbound peptide, and then removing the carrier.
  • the labeled antibody of the invention (7) is bound to the antibody + antigen peptide conjugate remaining above, and the signal of the labeled antibody is detected.
  • This method is a so-called “sandwich method”.
  • an enzyme used as a marker
  • the method is widely used as “ELISA enzyme linked immunosorbent assay”.
  • the two types of antibodies can both be monoclonal antibodies, or one of them can be a polyclonal antibody. Detection of the signal can be performed in the same manner as in the invention (8). Further, the diagnostic kit of the invention (17) is provided so as to enable simple and wide-ranging implementation of such a diagnostic method.
  • the diagnostic kits of Inventions (13) to (17) implement the diagnostic methods of Inventions (8) to (11). It is a reagent kit for use. Various types of such kits are commercially available depending on the type of the test component.
  • the diagnostic kits of the present invention also include the antigen peptides, antibodies, and labeled antibodies provided by the present invention. Except for the use of a publicly-available kit, it can be composed of the components used in a publicly-available kit.
  • the diagnostic method of the invention (12) is a method for testing the amount of the polynucleotide of the invention (3) in a biological sample of a subject, and determining whether the subject has a greater amount of the polynucleotide than that of a healthy subject. Or, it is judged as a high risk person.
  • the abundance of the polynucleotide of the subject is 10% or more, preferably 30 ° /, as compared with that of the healthy subject. As mentioned above, it is more preferably 70% or more, most preferably 100% or more.
  • the biological sample may be stool, blood, or blood cells (such as lymphocytes).
  • Polynucleotide detection and measurement can be performed by a known PCR method, RT-PCR method, quantitative RT-PCR method, or the like. In that case, the primer set of the invention (5) can be used for PCR.
  • the diagnostic method of the invention (12) can be carried out by a DNA microarray provided with the polynucleotide or the oligonucleotide provided by the present invention.
  • a method for preparing a microarray a method of directly synthesizing an oligonucleotide on the surface of a solid support (on-chip method) and a method of immobilizing a previously prepared oligonucleotide on the surface of the solid support are known.
  • the microarray used in the present invention can be produced by any of these methods.
  • the on-chip method combines the use of protective groups that are selectively removed by light irradiation with the photolithography technology and solid-phase synthesis technology used in semiconductor manufacturing, to achieve the specified fine-grained matrix. Selective synthesis in a region (masking technique: for example, Fodor, SPA Science 251: 767, 1991) it can.
  • the oligonucleotide with the functional group introduced is synthesized, and the oligonucleotide is spotted on the surface of the surface-treated solid support to form a covalent bond.
  • Oligonucleotides are generally covalently bonded to a surface-treated solid support via spacer crosslinkers.
  • a method is also known in which polyacrylamide gel micro-pieces are aligned on a glass surface and a synthetic oligonucleotide is covalently bonded thereto (Yershov, G. et al. Proc. Natl. Acad. Sci. USA 94: 4913, 1996).
  • an array of microelectrodes was prepared on a silica microarray, an agarose infiltration layer containing streptavidin was provided on the electrode to serve as a reaction site, and this site was positively charged to allow the biotinylated oligonucleotide to be charged. It is also known to enable fast and strict hybridization by fixing and controlling the charge of the site (Sosnowski, RG et al. Proc. Natl. Acad. Sci. USA 94 : 1119-1123, 1997).
  • cDNA is synthesized and PCR amplified, for example, by using mRNA isolated from the cells of the subject as type III.
  • the labeled dNTPs are incorporated into a labeled cDNA.
  • the labeled cDNA is brought into contact with the macroarray to detect the cDNA hybridized to the microarray capture probe (oligonucleotide or polynucleotide).
  • Hybridization can be carried out by dispensing a 96-well or 384-well plastic plate and applying an aqueous solution of the labeled cDNA on a microphone array.
  • the amount of spotting can be on the order of l-100 nl.
  • the hybridization is preferably performed at a temperature in the range of room temperature to 70 ° C. for 6 to 20 hours. After the hybridization, use a mixed solution of surfactant and buffer.
  • wash to remove unreacted labeled cDNA It is preferable to use sodium dodecyl sulfate (SDS) as the surfactant.
  • SDS sodium dodecyl sulfate
  • As the buffer a citrate buffer, a phosphate buffer, a borate buffer, a Tris buffer, a good buffer, and the like can be used, but a citrate buffer is preferably used.
  • the method for treating or preventing vasculitis according to the invention (19) is a method of removing blood from a patient, selectively removing anti-human peroxyredoxin antibody in the blood, and then returning the blood to the patient again.
  • the method according to the invention (19) is characterized in that “(a) a blood peroxyredoxin or an antigenic determinant thereof obtained by immobilizing blood or a fraction thereof containing antibodies collected from a patient on a carrier insoluble in blood. (Hereinafter referred to as “contact step”) and “(b) returning the blood or a fraction thereof to the patient after step (a)”.
  • the present inventors have confirmed for the first time that the serum of a patient with vasculitis has a significantly high anti-hyperoxyredoxin antibody.
  • the results suggest that anti-hyperperoxyredoxin antibodies are strongly associated with the development or progression of vasculitis. Therefore, it is considered that the elimination of anti-hyperoxyredoxin antibody in blood can improve or prevent the symptoms of vasculitis.
  • the anti-human peroxyredoxin antibody is adsorbed by the immobilized human peroxyredoxin or a fragment containing the antigenic determinant thereof in the contacting step. The antibodies are removed from the blood.
  • Blood taken from a patient may be subjected to treatment to separate it into several fractions before contacting with human peroxyredoxin or the like.
  • blood is separated into a plasma fraction and a cell fraction by a plasma separator, and only the plasma fraction in which an anti-hydroxy redoxin antibody is present is subjected to the contacting step. it can.
  • Plasma from which anti-human peroxyredoxin antibodies have been removed It is returned to the patient's body together with the cell fraction.
  • the therapeutic agent of the invention (18) can be used.
  • the therapeutic agent of the invention (18) contains human peroxyredoxin or a fragment containing the antigenic determinant thereof in a state immobilized on a blood-insoluble carrier. Therefore, when blood extracted from a patient is passed through the therapeutic agent, it is possible to selectively remove the anti-hyperperoxyredoxin antibody in the blood.
  • a carrier insoluble in blood cellulose, agarose, sepharose, dextran, chitin, chitosan, derivatives thereof, organic or inorganic porous materials, magnetic beads, microbeads, etc. can be used. It is not limited to these as long as they are insoluble.
  • the therapeutic agent of the invention (18) can be obtained by immobilizing a fragment containing a hyperperoxyredoxin or an antigenic determinant thereof on the carrier by physical adsorption or insolubilization by covalent bond.
  • Figure la is a photograph of a cell extracted protein separated and developed by two-dimensional electrophoresis and visualized by Coomassie staining.
  • Figure lb is a photograph of the developed protein transferred to a nitrocellulose membrane and reacted with the sera of four patients with vasculitis.
  • FIG. 2 is a photograph showing the results of one-dimensional electrophoresis of MBP and the fusion protein MBP-BC452 and staining with peroxidase-labeled Ni-NTA.
  • FIG. 3 is a photograph showing the results of a diagnosis of vasculitis by ELISA in which recombinant BC000452 was immobilized as an antigen peptide.
  • a protein corresponding to the positive protein spot 28 identified in FIG. Lb was excised and recovered from the gel in FIG. La, and extracted from the gel by trypsin digestion.
  • the mass of this digested peptide was determined by the mass-fingerprinting method using a time-of-flight mass spectrometer, and a candidate protein having a matching mass was selected from a protein database search using search software (mascot).
  • this antigen peptide was a PRDX2 homolog (GenBank / BC000452) (hereinafter, this peptide may be referred to as “BC452”).
  • Example 1 To confirm that the BC452 identified in Example 1 was truly an antigen that reacts with patient autoantibodies, a recombinant BC452 was created.
  • RT-PCR from mRNA prepared from umbilical vein-derived vascular endothelial cells
  • the coding region of BC452 cDNA (SEQ ID NO: 1) was amplified, cloned into E. coli protein expression vector (pMAL-cHis), introduced into E. coli, and expressed as a fusion protein with maltose binding protein (MBP) .
  • E. coli protein expression vector pMAL-cHis
  • MBP maltose binding protein
  • Figure 2a shows the results of purification of MBP (control) and fusion protein MBP-BC452, followed by one-dimensional electrophoresis and staining with Ni-NTA that recognizes the histidine tag located at the C-terminal of each protein. Yes, production and purification of each protein were confirmed.
  • MBP and the fusion protein MBP-BC452 were developed in the same lane and reacted individually with sera of various vasculitis patients. The results are shown in Figure 2b.
  • the upper band (fusion protein MBP-BC452) showed a much stronger staining than the S-MBP, indicating that autoantibodies against BC452 were present in the serum of patients with various vasculitis patients. Confirmed to be present.
  • the recombinant BC452 prepared in Example 2 was immobilized as an antigen peptide, reacted with a test serum, and further reacted with a peroxidase-labeled anti-human IgG antibody, and a peroxidase substrate was added to develop a color. Binding to the Seichu antibody was detected.
  • vasculitis Pulmona monogranulomatosis, microscopic polyangiitis, allergic granulomatous vasculitis, polyarteritis nodosa, temporal arteritis, Takayasu disease, rheumatoid arthritis, Bajaja disease Sera from systemic lupus erythematosus), patients with collagen disease without vasculitis, and healthy individuals.
  • Figure 3 is a numerical representation of the color reaction as a 0D value.
  • the value obtained by adding three times the standard deviation to the mean 0D value of a healthy person was defined as 100 units, and sera with a higher number of units were determined to be positive.
  • Autoantibodies to BC452 were detected in 68% of patients with vasculitis, 15% in patients with collagen disease without vasculitis, healthy was 0%. From these results, it was confirmed that the detection of an autoantibody to the antigen peptide BC452 is a very useful marker for diagnosing vasculitis.
  • the invention of this application provides a novel antigenic peptide useful as a diagnostic marker for vasculitis and a method for diagnosing vasculitis using the same. This enables early and highly accurate diagnosis of vasculitis.

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Abstract

A human vasculitis antigen peptide which is a peroxiredoxin; and a method of diagnosing vasculitis which comprises examining the presence or absence of an antibody binding to the above antigen peptide in the serum of a subject.

Description

明細書 血管炎抗原べプチドと血管炎診断方法 技術分野  Description Vasculitis antigen peptide and vasculitis diagnostic method
この出願の発明は、 ヒ ト血管炎の抗原ペプチドと、 このペプチドをコ ードするポリヌクレオチド、 並びにこれらを用いた血管炎診断方法に関 するものである。 背景技術  The invention of this application relates to an antigen peptide for human vasculitis, a polynucleotide encoding the peptide, and a method for diagnosing vasculitis using the same. Background art
結節性多発動脈炎などの血管炎症を主体とする一群の疾患 （いわゆる 血管炎症候群） は、 一般に難治性かつ予後不良であり、 その病因解明が 社会的要請となっている。 血管炎に含まれる疾患として、 結節性多発動 脈炎の他、 ゥェゲナー肉芽腫症、 顕微鏡的結節性多発血管炎、 アレルギ 一性肉芽腫性血管炎、 結節性多発動脈炎、 側頭動脈炎、 高安動脈炎、 悪 性関節リ ウマチ、 バージャ一病、 抗リ ン脂質抗体症候群、 川崎病、 へノ ッホ-シェンライン紫斑病、 リベドー紫斑病、 半月体形成腎炎、 肺毛細 血管炎、 チヤーグ-ス トラウス症候群等、 多くの疾病が知られている。 また、 皮膚筋炎、 混合性結合組織病、 強皮症、 ベーチェッ ト病、 シエー グレン症候群、 全身性エリテマトーデスなどの膠原病において血管炎を 合併しうることが知られている。 発症の機序としては自己免疫機序の関 与が考えられているが、 その詳細は不明である。  A group of diseases mainly composed of vascular inflammation such as polyarteritis nodosa (so-called vasculitis syndrome) is generally intractable and has a poor prognosis, and elucidation of the etiology is a social demand. Diseases that are included in vasculitis include polygenetic nodular arthritis, Zegener's granulomatosis, microscopic polyarteritis nodosa, allergic granulomatous vasculitis, polyarteritis nodosa, temporal arteritis, Takayasu's arteritis, rheumatoid arthritis, Baja disease, anti-phospholipid antibody syndrome, Kawasaki disease, Henoch-Schenlein purpura, Libedo purpura, crescentic nephritis, pulmonary capillary vasculitis, chiag- Many diseases are known, such as Strauss syndrome. It is also known that vasculitis can be associated with collagen diseases such as dermatomyositis, mixed connective tissue disease, scleroderma, Behcet's disease, Siegren's syndrome, and systemic lupus erythematosus. An autoimmune mechanism is thought to be involved in the pathogenesis, but the details are unknown.
一方、 各種のヒ ト疾患に対して、 その疾患に特異的な血清マーカーを 指標とする分子生物学的診断が普及しつつある。 この方法は、 抗体と反 応性を有する患者血清中の自己抗原ペプチドの存在、 または抗原べプチ ドに反応する自己抗体の存在を検出するものであり、 大がかりな設備を 必要とせず、 被験者への負担も少ないため、 自覚症状のない多くの被験 者に対しても広範囲に実施することが可能である。 血管炎においても、 例えば ANCA等の診断的価値のある自己抗体が報告されているが （Bartu nkova J, Tesar V, sediva A. Diagnostic and pathogenetic rol e of ant ineutrophi l cytoplasmi c autoant ibodi es. Cl in Immunol 2003； 10 6 : 73-82)、 ANCA 陰性の血管炎も多く あり、 診断指標として不十分であ る。 発明の開示 On the other hand, for various human diseases, molecular biological diagnosis using serum markers specific to the disease as an index is becoming widespread. This method detects the presence of an autoantigen peptide in the serum of a patient who has reactivity with an antibody, or the presence of an autoantibody that reacts with an antigen peptide. Since it is not required and the burden on the subjects is small, it can be widely performed for many subjects without subjective symptoms. In vasculitis, for example, autoantibodies of diagnostic value such as ANCA have been reported (Bartu nkova J, Tesar V, sediva A. Diagnostic and pathogenetic rol e of ant ineutrophi l cytoplasmic c autoant ibodi es. Immunol 2003; 106: 73-82), and there are many ANCA-negative vasculitis, which is not sufficient as a diagnostic index. Disclosure of the invention
この出願の発明は、 以上のとおりの事情に鑑みてなされたものであつ て、 血管炎の診断に有効な新規抗原ぺプチドを提供することを課題とし ている。  The invention of this application has been made in view of the circumstances described above, and has an object to provide a novel antigen peptide which is effective for diagnosing vasculitis.
またこの出願の発明は、 前記の抗原べプチドをコ一ドする遺伝子材料 と、 抗原ぺプチドに対する抗体を提供することを課題としている。  It is another object of the invention of this application to provide a genetic material encoding the above antigen peptide and an antibody against the antigen peptide.
さらにこの出願の発明は、 前記のペプチド、 ポリヌクレオチドおよび 抗体等を用いた血管炎診断方法を提供することを課題としている。  It is a further object of the invention of the present application to provide a method for diagnosing vasculitis using the above-mentioned peptide, polynucleotide, antibody and the like.
この出願は、 前記の課題を解決するものとして、 以下の（1)〜（17)の 発明を提供する。  This application provides the following inventions (1) to (17) to solve the above problems.
(1) ヒ ト ' ペルォキシレドキシンである血管炎抗原べプチド。  (1) Human 'peroxiredoxin, a vasculitis antigen peptide.
(2) 配列番号 2のアミノ酸配列を有するヒ ト ·ペルォキシレドキシン 2 相同体である前記発明（1)の血管炎抗原べプチド。 (2) The vasculitis antigen peptide according to the invention (1), which is a human peroxyredoxin 2 homolog having the amino acid sequence of SEQ ID NO: 2.
(3) 前記発明（1)の抗原べプチドをコードするポリヌクレオチド。  (3) A polynucleotide encoding the antigenic peptide of the invention (1).
(4) 配列番号 1の塩基配列を有し、 前記発明（2)のヒ ト · ペルォキシレ ドキシン 2相同体をコードする前記発明（3)のポリヌクレオチド。 (5) 前記発明（3)のポリヌクレオチドを PCR 増幅するためのプライマー セッ ト。 (6) 前記発明（1 )の抗原ペプチドと結合する抗体。 (4) The polynucleotide according to the invention (3), which has the nucleotide sequence of SEQ ID NO: 1 and encodes the human peroxyredoxin 2 homologue according to the invention (2). (5) A primer set for PCR-amplifying the polynucleotide of the invention (3). (6) An antibody that binds to the antigenic peptide of the invention (1).
(7) 前記発明（6)の抗体とは異なるェピトープと結合する抗体。  (7) An antibody that binds to an epitope different from the antibody of the invention (6).
(8) 被験者から採取した生体試料中の前記発明（1 )の抗原べプチドと結 合する抗体を検出し、 生体試料中にその抗体が健常者よりも多く存 在する被験者を血管炎患者または血管炎ハイリスク者と判定するこ とを特徴とするヒ ト血管炎診断方法。  (8) An antibody that binds to the antigenic peptide of the invention (1) is detected in a biological sample collected from a subject, and a subject in which the antibody is present in a biological sample in a greater amount than a healthy subject is identified as a vasculitis patient or A method for diagnosing human vasculitis, characterized by determining that the patient has a high risk of vasculitis.
(9) 抗原べプチドを固定化した担体上において被験者から採取した生体 試料中の抗体と抗原べプチドとの結合を試験する前記発明（8)の診 断方法。  (9) The diagnostic method according to the invention (8), wherein the binding between the antibody in the biological sample collected from the subject and the antigen peptide is tested on a carrier having the antigen peptide immobilized thereon.
(10) 被験者から採取した生体試料中の前記発明（6)の抗体と結合する抗 原ぺプチドを検出し、 生体試料中にその抗原べプチドが健常者よ り も多く存在する被験者を血管炎患者または血管炎ハイ リスク者 と判定することを特徴とするヒ ト血管炎診断方法。 (10) An antigen peptide that binds to the antibody of the invention (6) is detected in a biological sample collected from a subject, and the subject whose antigen peptide is present in the biological sample in a greater amount than in a healthy subject is subjected to vasculitis. A method for diagnosing human vasculitis, which is determined to be a patient or a person at high risk for vasculitis.
(1 1 ) 前記発明（6)の抗体を固定化した担体上において、 抗体と抗原ぺプ チドの結合を試験する前記発明（10)の診断方法。  (11) The diagnostic method according to the invention (10), wherein the binding between the antibody and the antigen peptide is tested on a carrier on which the antibody according to the invention (6) is immobilized.
( 12) 被験者から採取した生体試料における前記発明（3)のポリヌクレオ チドの存在量を試験し、 ポリヌクレオチドの存在量が健常者のそ れらと比較して多い被験者を血管炎患者または血管炎ハイ リスク 者と判定することを特徴とするヒ ト血管炎診断方法。  (12) Examining the amount of the polynucleotide of the invention (3) in a biological sample collected from a subject, and determining whether the subject has a higher polynucleotide abundance as compared to those of a healthy subject. A method for diagnosing human vasculitis, characterized by determining that the subject is at high risk.
(13) 少なく とも以下の要素 ： (13) At least the following elements:
(a) 前記発明（1 )の抗原ペプチド； および  (a) the antigen peptide of the invention (1); and
(b) 前記発明（1 )の抗原べプチドと結合する抗体に特異的に結合 する標識化抗体  (b) a labeled antibody that specifically binds to the antibody that binds to the antigenic peptide of the invention (1)
からなることを特徴とする血管炎診断キッ ト。  A diagnostic kit for vasculitis, comprising:
( 14) 少なく とも以下の要素 ： (14) At least the following elements:
(a) 前記発明（1)の抗原べプチドを固定化した担体；および (b) 前記発明（1 )の抗原ぺプチドと結合する抗体に特異的に結合 する標識化抗体 (a) a carrier on which the antigen peptide of the invention (1) is immobilized; and (b) a labeled antibody that specifically binds to the antibody that binds to the antigen peptide of the invention (1)
からなることを特徴とする血管炎診断キッ ト。  A diagnostic kit for vasculitis, comprising:
(15) 少なく とも、 前記発明（6)の抗体および/または標識化した前記発 明（7)の抗体を含むことを特徴とする血管炎診断キッ ト。  (15) A diagnostic kit for vasculitis comprising at least the antibody of the invention (6) and / or the labeled antibody of the invention (7).
(16) 少なく とも以下の要素 ：  (16) At least the following elements:
(a) 前記発明（6)の抗体； および  (a) the antibody of the invention (6); and
(b) 標識化した前記発明（7)の抗体  (b) the labeled antibody of the invention (7)
からなることを特徴とする血管炎診断キッ ト。  A diagnostic kit for vasculitis, comprising:
( 17) 少なく とも以下の要素 ： (17) At least the following elements:
(a) 前記発明（6)の抗体を固定化した担体； および  (a) a carrier on which the antibody of the invention (6) is immobilized; and
(b) 標識化した前記発明（7)の抗体  (b) the labeled antibody of the invention (7)
からなることを特徴とする血管炎診断キッ ト。  A diagnostic kit for vasculitis, comprising:
( 18) 血液に不溶性の担体に固定化されたヒ トのペルォキシレドキシン またはその抗原決定基を含む断片を含む、 血管炎の治療剤。  (18) A therapeutic agent for vasculitis, comprising human peroxyredoxin or a fragment containing an antigenic determinant thereof, which is immobilized on a carrier insoluble in blood.
(19) 次の工程を含む、 血管炎の治療または予防方法。  (19) A method for treating or preventing vasculitis, comprising the following steps.
(a) 患者から採取した血液または抗体を含むその分画を、 血液に 不溶性の担体に固定化されたヒ トのペルォキシレドキシン またはその抗原決定基を含む断片に接触させる工程、 およ び  (a) contacting blood or a fraction thereof containing antibodies collected from a patient with human peroxyredoxin or a fragment thereof containing an antigenic determinant immobilized on a blood-insoluble carrier; and And
(b) 工程（a)の後に当該血液またはその分画を、 患者に戻す工程。 (b) returning the blood or a fraction thereof to the patient after step (a).
(20) 抗体を含む分画が血漿である前記発明（19)に記載の方法。 (20) The method according to the above-mentioned invention (19), wherein the fraction containing the antibody is plasma.
すなわち、 この出願の発明者らは、 2次元電気泳動法 （例えば、 Elec trophores i s 22 : 3019-3025, 2001 ) により血管内皮細胞に特異的な自己 抗原ペプチドを網羅的に解析することによって、 血管炎患者血清が反応 する血管炎抗原べプチドを同定し、 それがペルォキシレドキシンまたは チォレドキシンペルォキシダーゼであることを見出してこの発明を完成 させた。 ペルォキシレドキシンまたはチォレドキシンペルォキシダーゼ はその抗酸化作用や、 酸素ス トレスに対する細胞反応等に関係する酵素 タンパク質として知られているが、 血管炎との関係、 特に血管炎患者血 清中の抗体と特異的に反応することは、 従来全く知られていない。 That is, the inventors of the present application analyzed vascular endothelial cells by comprehensive analysis of autoantigen peptides specific to vascular endothelial cells by two-dimensional electrophoresis (for example, Elec trophores is 22: 3019-3025, 2001). Identified the vasculitis antigen peptide to which the sera of patients with inflammation reacted, which was identified as peroxyredoxin or The present invention was completed by finding that it was thioredoxin peroxidase. Peroxyredoxin or thioredoxin peroxidase is known as an enzyme protein related to its antioxidant activity and cellular response to oxygen stress, etc., but it has a relationship with vasculitis, especially in the plasma of patients with vasculitis. It has never been known that it reacts specifically with the antibody in it.
この出願の発明は、 以上のとおりの新規抗原ペプチドと、 そのべプチ ドに対する抗体、 それらをコードするポリヌクレオチドを基礎とするも のである。  The invention of this application is based on the novel antigenic peptide described above, an antibody against the peptide, and a polynucleotide encoding the same.
なお、 この発明において、 「タンパク質」 および 「ペプチド」 とは、 アミ ド結合 （ペプチド結合） によって互いに結合した複数個のアミノ酸 残基から構成された分子を意味する。 「ポリヌクレオチド」 とは、 プリ ンまたはピリ ミジンが糖に - N -ダリコシド結合したヌクレオシドのリ ン酸エステル （ATP、 GTP、 CTP、 UTP； または dATP、 dGTP、 dCTP、 dUT P) が 100個以上結合した分子を言い、 「オリゴヌクレオチド」 とは 2 - 9 9個連結した分子を言う。  In the present invention, “protein” and “peptide” mean a molecule composed of a plurality of amino acid residues linked to each other by an amide bond (peptide bond). “Polynucleotide” is a nucleoside phosphoric acid ester (ATP, GTP, CTP, UTP; or dATP, dGTP, dCTP, dUTP) containing at least 100 N-daricoside-linked pyridine or pyrimidine. "Oligonucleotide" refers to a linked molecule of 2-99.
また、 配列表に示した塩基配列およびアミノ酸配列については、 1以 上の塩基の付加、 欠失、 他の塩基への置換、 あるいはこれらの塩基変異 に基づく 1以上のアミノ酸残基の付加、 欠失おょぴ他のアミノ酸への置 換をも包含するものである。  Regarding the base sequence and amino acid sequence shown in the sequence listing, addition or deletion of one or more bases, substitution with other bases, or addition or deletion of one or more amino acid residues based on these base mutations It also includes substitution for other amino acids.
さらに、 「ペルォキシレドキシンを認識する抗体」 とは、 発明（1 )の抗 原べプチドと結合する抗体を意味するが、 ペルォキシレドキシンと非特 異的に結合する抗体は含まない。 また、 発明（6)および（7)の 「抗体」 は、 発明（1 )の抗原ぺプチドを免疫原として作製されたポリクローナル抗体 またはモノクローナル抗体を意味する。  Further, “an antibody recognizing peroxyredoxin” means an antibody that binds to the antigen peptide of the invention (1), but does not include an antibody that binds nonspecifically to peroxyredoxin. . The “antibody” of the inventions (6) and (7) means a polyclonal antibody or a monoclonal antibody prepared using the antigen peptide of the invention (1) as an immunogen.
この発明におけるその他の用語や概念は、 発明の実施形態の説明や実 施例において詳しく規定する。 またこの発明を実施するために使用する 様々な技術は、 特にその出典を明示した技術を除いては、 公知の文献等 に基づいて当業者であれば容易かつ確実に実施可能である。 例えば、 こ の発明の診断方法等に使用可能な薬剤の調製は Remington' s Pharmaceu t ical Sciences, 18th Edit ion, ed. A. Gennaro, Mack Publ i shing C o. , Easton, PA, 1990に、 遺伝子工学および分子生物学的技術は Sambr ook and Maniati s, in Molecular Cloning - A Laboratory Manual, ColdOther terms and concepts in the present invention are defined in detail in the description of the embodiments of the present invention and examples. Also used to practice this invention Various techniques can be easily and reliably implemented by those skilled in the art based on known documents and the like, except for techniques for which the source is clearly indicated. For example, the preparation of a drug that can be used in the diagnostic method of the present invention is described in Remington's Pharmaceutical Sciences, 18th Edition, ed.A. Gennaro, Mack Publishing Co., Easton, PA, 1990. Genetic engineering and molecular biology techniques are described in Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold
Spring Harbor Laboratory Press, New York, 1989 ; Ausubel, F. M. et al. , Current Protocol s in Molecular Biology, J ohn Wi ley & Son s, New York, N. Y， 1995等に記載されている。 Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995, and the like.
以下、 各発明について、 実施形態を詳しく説明する。  Hereinafter, embodiments of each invention will be described in detail.
発明（1)の血管炎抗原ペプチドは、 ペルォキシレドキシン （Peroxired oxin： PRDX) である。 この PRDX はまた、 チォレドキシンペルォキシダ ーゼ ( Thioredoxin peroxi dase) と呼称されることもある。 PRDX とし ては、 PRDX1 ( GenBank/NM_002574) , PRDX2 (GenBank/NM— 005809)、 PRDX 3 (GenBank/丽— 006793)、 PRDX4 (GenBank/匪— 006406)、 PRDX5 (GenBank /NM— 012094)、 PRDX6 ( GenBank/NM— 004905) 力 S公知である。 またチ才レ ドキシンペルォキシダーゼとしては GenBank/BC003609、 GenBank/BC007 107 等が公知である。 さらに、 PRDX2 (チォレドキシンペルォキシダー ゼ 1) の相同体としては GenBank/BC003022、 GenBank/BC000452 が公知 である。 従って、 この発明（1)の血管炎抗原はこれら公知の PRDXを全て 包含するが、 特に配列番号 2のアミノ酸配列を有する PRDX2相同体 （Ge nBank/BC000452) が好ましい （発明（2) )。  The vasculitis antigen peptide of the invention (1) is Peroxired oxin (PRDX). This PRDX may also be referred to as Thioredoxin peroxidase. PRDX includes: PRDX1 (GenBank / NM_002574), PRDX2 (GenBank / NM—005809), PRDX 3 (GenBank / 丽 —006793), PRDX4 (GenBank / Martian—006406), PRDX5 (GenBank / NM—0112094), PRDX6 (GenBank / NM—004905) Force S Known. GenBank / BC003609, GenBank / BC007 107, etc. are known as red-red redoxin peroxidase. Further, as homologues of PRDX2 (thioredoxin peroxidase 1), GenBank / BC003022 and GenBank / BC000452 are known. Therefore, the vasculitis antigen of the present invention (1) includes all of these known PRDXs, but a PRDX2 homolog (GenBank / BC000452) having the amino acid sequence of SEQ ID NO: 2 is particularly preferable (the invention (2)).
これらの抗原ペプチド （PRDX) は、 例えば、 発明（3)のポリヌクレオ チドを保有する組換え発現ベクターからインビトロ転写によって RNAを 調製し、 これを鎵型としてインビトロ翻訳を行うことによりインビトロ でペプチドを発現できる。 また組換え発現ベクターを大腸菌、 枯草菌等 の原核細胞や、 酵母、 昆虫細胞、 哺乳動物細胞等の真核細胞に導入して 形質転換細胞を作製すれば、 この形質転換細胞からぺプチドを発現させ ることができる。 These antigenic peptides (PRDX) can be expressed, for example, by preparing RNA from the recombinant expression vector having the polynucleotide of the invention (3) by in vitro transcription and performing in vitro translation using this as a type III to express the peptide in vitro. it can. E. coli, Bacillus subtilis, etc. When transfected cells are introduced into eukaryotic cells such as prokaryotic cells, eukaryotic cells such as yeast cells, insect cells, and mammalian cells to produce transformed cells, peptides can be expressed from the transformed cells.
抗原べプチドをインビト口翻訳で発現させる場合には、 ポリヌクレオ チドを、 RNA ポリメラーゼプロモーターを有するベクターに揷入して組 換え発現ベクターを作製し、 このベクターを、 プロモーターに対応する RNA ポリメラーゼを含むゥサギ網状赤血球溶解物や小麦胚芽抽出物など のィンビトロ翻訳系に添加すれば、 抗原べプチドをィンビ トロで生産す ることができる。 RNA ポリメラーゼプロモーターとしては、 T7、 T3、 SP 6などが例示できる。 これらの RNAポリメラーゼプロモーターを含むベ クタ一としては、 pKAl、 pCDM8、 pT3/T7 18、 ρΤ7/3 19、 pBluescript I I などが例示できる。  When expressing the antigen peptide by in vitro translation, the polynucleotide is inserted into a vector having an RNA polymerase promoter to prepare a recombinant expression vector, and this vector is used as a rabbit containing the RNA polymerase corresponding to the promoter. When added to an in vitro translation system such as a reticulocyte lysate or a wheat germ extract, the antigen peptide can be produced in vitro. Examples of the RNA polymerase promoter include T7, T3, and SP6. Examples of vectors containing these RNA polymerase promoters include pKAl, pCDM8, pT3 / T718, ρΤ7 / 319, pBluescript II and the like.
抗原ペプチドを、 大腸菌などの微生物で発現させる場合には、 微生物 中で複製可能なオリ ジン、 プロモーター、 リボソーム結合部位、 DNA ク ローニング部位、 ターミネータ一等を有するベクターにポリヌクレオチ ドを組換えた発現ベクターを作製し、 この発現ベクターで宿主細胞を形 質転換したのち、 得られた形質転換体を培養すれば、 そのポリヌク レオ チドがコードしている抗原べプチドを微生物から発現させることができ る。 この際、 他のタンパク質との融合タンパク質として発現させること もできる。 大腸菌用発現ベクターとしては、 pUC 系、 pBluescript I I、 pET 発現システム、 pGEX 発現システム、 pMAL 発現システムなどが例示 できる。  When expressing antigen peptides in microorganisms such as Escherichia coli, expression is performed by recombining polynucleotides into a vector having an origin, a promoter, a ribosome binding site, a DNA cloning site, a terminator, etc. that can be replicated in microorganisms. After preparing a vector, transforming a host cell with this expression vector, and culturing the resulting transformant, the antigen peptide encoded by the polynucleotide can be expressed from the microorganism. . At this time, it can be expressed as a fusion protein with another protein. Examples of expression vectors for Escherichia coli include a pUC system, pBluescript II, a pET expression system, a pGEX expression system, and a pMAL expression system.
抗原ペプチドを、 真核細胞で発現させる場合には、 ポリヌクレオチド を、 プロモーター、 スプライシング領域、 ポリ （A)付加部位等を有する 真核細胞用発現ベクターに挿入して組換えベクターを作製し、 真核細胞 内に導入すれば、 抗原べプチドを形質転換真核細胞で発現させることが できる。 発現ベクターとしては、 pKAl、 pCDM8、 pSVK3、 pMSG、 PSVL、 B K - CMV、 pBK RSV、 EBVベクター、 pRS、 pcDNA3、 pMSG、 pYES2など力 S例示 できる。 また、 pIND/V5 - Hi s、 pFLAG-CMV- 2、 pEGFP- Nl、 pEGFP - CI など を発現ベクターとして用いれば、 Hi sタグ、 FLAGタグ、 mycタグ、 HAタ グ、 GFP など各種タグを付加した融合タンパク質として抗原ペプチドを 発現させることもできる。 真核細胞と しては、 サル腎臓細胞 C0S7、 チ ャィニーズハムスター卵巣細胞 CH0などの哺乳動物培養細胞、 出芽酵母、 分裂酵母、 カイコ細胞、 アフリカッメガエル卵細胞などが一般に用いら れるが、 この発明の抗原ペプチドを発現できるものであれば、 いかなる 真核細胞でもよい。 発現ベクターを真核細胞に導入するには、 電気穿孔 法、 リン酸カルシウム法、 リボソーム法、 DEAE デキス トラン法など公 知の方法を用いることができる。 When the antigen peptide is expressed in eukaryotic cells, the polynucleotide is inserted into an expression vector for eukaryotic cells having a promoter, a splicing region, a poly (A) addition site, etc. to prepare a recombinant vector. When introduced into nuclear cells, antigenic peptides can be expressed in transformed eukaryotic cells. it can. Expression vectors, pKAl, pCDM8, pSVK3, pMSG , P SVL, BK - CMV, pBK RSV, EBV vector, pRS, pcDNA3, pMSG, pYES2 such force S can be exemplified. If pIND / V5-His, pFLAG-CMV-2, pEGFP-Nl, pEGFP-CI, etc. are used as expression vectors, various tags such as His tag, FLAG tag, myc tag, HA tag, and GFP can be added. An antigen peptide can also be expressed as a fused protein. As eukaryotic cells, mammalian cultured cells such as monkey kidney cells C0S7 and Chinese hamster ovary cells CH0, budding yeast, fission yeast, silkworm cells, African egg cells and the like are generally used. Any eukaryotic cell can be used as long as it can express the antigenic peptide of the present invention. In order to introduce the expression vector into eukaryotic cells, known methods such as an electroporation method, a calcium phosphate method, a ribosome method, and a DEAE dextran method can be used.
抗原べプチドを原核細胞や真核細胞で発現させたのち、 培養物から目 的ぺプチドを単離精製するためには、 公知の分離操作を組み合わせて行 うことができる。 例えば、 尿素などの変性剤や界面活性剤による処理、 超音波処理、 酵素消化、 塩析ゃ溶媒沈殿法、 透析、 遠心分離、 限外濾過、 ゲル濾過、 SDS- PAGE、 等電点電気泳動、 イオン交換クロマトグラフィー、 疎水性クロマトグラフィー、 ァフィ二ティークロマトグラフィー、 逆相 クロマトグラフィーなどが挙げられる。  After expressing the antigen peptide in prokaryotic cells or eukaryotic cells, the target peptide can be isolated and purified from the culture by a combination of known separation procedures. For example, treatment with denaturing agents such as urea or surfactants, ultrasonic treatment, enzyme digestion, salting-out / solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, Examples include ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.
なお、 以上の方法によって得られる組換え抗原ペプチドには、 他の任 意のタンパク質との融合タンパク質も含まれる。 例えば、 グルタチオン 一 S—トランスフェラーゼ （GST) や緑色蛍光蛋白質 （GFP) との融合蛋 白質などが例示できる。 さらに、 形質転換細胞で発現されたペプチドは、 翻訳された後、 細胞内で各種修飾を受ける場合がある。 したがって、 修 飾されたペプチドも第 1発明の抗原ペプチドの範囲に含まれる。 このよ うな翻訳後修飾としては、 N末端メチォニンの脱離、 ァセチル化、 糖鎖 付加、 細胞内プロテアーゼによる限定分解、 ミ リ ス トイル化、 イソプレ ニル化、 リン酸化などが例示できる。 The recombinant antigen peptide obtained by the above method also includes a fusion protein with any other protein. For example, a fusion protein with glutathione-1S-transferase (GST) and green fluorescent protein (GFP) can be exemplified. Further, peptides expressed in transformed cells may undergo various modifications in the cells after translation. Therefore, the modified peptide is also included in the scope of the antigen peptide of the first invention. Such post-translational modifications include N-terminal methionine elimination, acetylation, and sugar chains. Examples thereof include addition, limited degradation by intracellular protease, myristoylation, isoprenylation, and phosphorylation.
以上の方法により得られた抗原べプチドは、 この発明によって提供さ れる血管炎診断方法の材料として使用される。  The antigen peptide obtained by the above method is used as a material for the method of diagnosing vasculitis provided by the present invention.
発明（3)は、 発明（1 )の抗原ペプチドをコードするポリヌク レオチ ド ( DNA 断片、 RNA 断片） である。 具体的には、 各ペプチド （タンパク 質） をコードするゲノム DNA、 ゲノム DNA から転写される mRNA、 mRNA から合成される cDNA である。 また、 2本鎖であっても 1 本鎖であって もよい。 さらに、 これらのゲノム DNAや mRNA、 cDNA のセンス鎖おょぴ アンチセンス鎖も含まれる。 またさらに、 ゲノム DNAの場合には、 その 発現制御領域 （プロモーター、 ェンハンサー、 サブレッサー領域） をも 含む。  Invention (3) is a polynucleotide (DNA fragment, RNA fragment) encoding the antigenic peptide of invention (1). Specifically, genomic DNA encoding each peptide (protein), mRNA transcribed from genomic DNA, and cDNA synthesized from mRNA. It may be double-stranded or single-stranded. Furthermore, the sense strand and antisense strand of these genomic DNA, mRNA and cDNA are also included. Furthermore, in the case of genomic DNA, it also includes its expression control region (promoter, enhancer, sublesser region).
これらのポリヌクレオチドは、 それぞれ公知の方法によって容易に取 得することができる。 例えば、 cDNA の場合には、 公知の方法 （Mol. Ce 11 Biol. 2， 161-170, 1982； J. Gene 25， 263 - 269, 1983； Gene, 15 0, 243-250, 1994) を用いて cDNA を合成し、 それぞれ公知の塩基配列 に基づいて作製したプローブ DNA を用いて、 それぞれの cDNA を単離す る方法によって取得することができる。 得られた cDNAは、 例えば、 PCR (Polymerase Chain Reaction) 法、 NASBA (Nuclei c aci d sequence ba sed ampl ificat ion) 法、 TMA (Transcription-mediated am l  These polynucleotides can be easily obtained by known methods. For example, in the case of cDNA, a known method (Mol. Ce 11 Biol. 2, 161-170, 1982; J. Gene 25, 263-269, 1983; Gene, 150, 243-250, 1994) is used. CDNA can be obtained by a method of isolating each cDNA using a probe DNA prepared based on a known base sequence. The obtained cDNA is subjected to, for example, PCR (Polymerase Chain Reaction), NASBA (Nucleic acid sequence base amplification), TMA (Transcription-mediated
if ication) 法おょぴ SDA ( Strand Di splacement Ampl if ication) 法な どの通常行われる遺伝子増幅法により增幅することができる。 また、 こ の発明によって提供されるプライマーセッ トを用い、 ヒ ト細胞から単離 した mRNAを铸型とする RT- PCR法によつても必要量の各 cDNAを得るこ とができる。 This method can be extended by a commonly used gene amplification method such as the SDA (Strand Displacement Amplification) method. Also, the required amount of each cDNA can be obtained by RT-PCR using the primer set provided by the present invention as a type II mRNA isolated from human cells.
このようにして取得されるポリヌクレオチドの好ましい態様は、 PRDX 2 相同体をコードする配列番号 1 の塩基配列を有するポリヌク レオチド (cDNA) である （発明（4))。 A preferred embodiment of the polynucleotide thus obtained is PRDX 2 Polynucleotide (cDNA) having the nucleotide sequence of SEQ ID NO: 1 encoding a homolog (invention (4)).
またさらに、 この発明（3)ポリヌクレオチドには、 その一部連続配列 からなるオリ ゴヌクレオチドも含まれる。 このようなオリゴヌクレオチ ドは、 例えば前記のポリヌク レオチ ド（cDNA)を適当な制限酵素で切断す ることによつても得ることができる。 あるいは、 Carruthers (1982) Co Id Spring Harbor Symp. Quant. Biol. 47:41ト 418; Adams (1983) J. Am. Chem. Soc. 105:661； Belousov (1997) Nucleic Acid Res. 25:344 0-3444； Frenkel (1995) Free Radio. Biol. Med. 19:373 - 380; Blomme rs (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetr a. Lett. 22： 1859； 米国特許第 4， 458， 066号に記載されているような周 知の化学合成技術により、 in vitro において合成することができる。 これらのオリ ゴヌクレオチドは、 例えば発明（1)のポリヌクレオチドを 単離するためのプローブとして使用することができる。 従って、 このォ リゴヌクレオチドは、 標識物質によって標識化されたものも含まれる。 標識は、 ラジオアイソ トープ （RI) 法または非 RI 法によって行うこと ができるが、 非 RI法 用いることが好ましい。 非 RI法としては、 蛍光 標識法、 ピオチン標識法、 化学発光法等が挙げられるが、 蛍光標識法を 用いることが好ましい。 蛍光物質としては、 オリゴヌクレオチドの塩基 部分と結合できるものを適宜に選択して用いることができるが、 シァニ ン色素 （例えば、 Cy Dye TMシリーズの Cy3、 Cy5等）、 ローダミ ン 6G試 薬、 N-ァセ トキシ- N2 -ァセチルァミ ノフルオレン （AAF)、 AAIF (AAF の ヨウ素誘導体） などを使用することができる。 Furthermore, the polynucleotide of the present invention (3) also includes an oligonucleotide consisting of a partially continuous sequence thereof. Such an oligonucleotide can also be obtained, for example, by cleaving the above-mentioned polynucleotide (cDNA) with an appropriate restriction enzyme. Or Carruthers (1982) Co Id Spring Harbor Symp. Quant. Biol. 47:41 to 418; Adams (1983) J. Am. Chem. Soc. 105: 661; Belousov (1997) Nucleic Acid Res. 25: 344 0 -3444; Frenkel (1995) Free Radio. Biol. Med. 19: 373-380; Blomme rs (1994) Biochemistry 33: 7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetr a. Lett. 22: 1859; can be synthesized in vitro by known chemical synthesis techniques as described in US Pat. No. 4,458,066. it can. These oligonucleotides can be used, for example, as probes for isolating the polynucleotide of the invention (1). Therefore, the oligonucleotide includes those labeled with a labeling substance. Labeling can be performed by a radioisotope (RI) method or a non-RI method, but it is preferable to use a non-RI method. Examples of the non-RI method include a fluorescent labeling method, a biotin labeling method, and a chemiluminescent method, and it is preferable to use a fluorescent labeling method. As the fluorescent substance, it can be used to select those that can bind with a base portion of the oligonucleotides appropriate, Shiani emissions dyes (e.g., Cy Dye TM series of Cy3, Cy5, etc.), Rodami down 6G reagent, N -Acetoxy-N 2 -acetylaminofluorene (AAF), AAIF (iodine derivative of AAF) and the like can be used.
発明（5)は、 発明（3)のポリヌクレオチドを PCR增幅するためのプライ マーセッ トであり、 これはそれぞれ公知の塩基配列に基づき設計し、 合 成 '精製の各工程を経て調製することができる。 なお、 プライマー設計 の留意点として、 例えば以下を指摘することができる。 プライマーのサ ィズ （塩基数） は、 鎵型 DNAとの間の特異的なアニーリングを満足させ ることを考慮し、 15- 40塩基、 望ましくは 15- 30塩基である。 ただし、 LA ( long accurate) PCRを行う場合には、 少なく とも 30塩基が効果的 である。 センス鎖 （5，末端側） とアンチセンス鎖 （3，末端側） からなる 1組あるいは 1対 （2本） のプライマーが互いにァニールしないよう、 両プライマー間の相捕的配列を避けると共に、 プライマー内のヘアピン 構造の形成を防止するため自己相捕配列をも避けるようにする。 さらに. 铸型 DNA との安定な結合を確保するため GC含量を約 50%にし、 プライ マー内において GC- ri chあるいは AT- ri chが偏在しないようにする。 ァ ニーリ ング温度は Tm (melt ing temperature) に依存するので、 特異性 の高い PCR産物を得るため、 Tm値が 55- 65°Cで互いに近似したプライマ 一を選定する。 また、 PCR におけるプライマー使用の最終濃度が約 0. 1 〜約 1 μ Μ になるよう調整する等を留意することも必要である。 また、 プライマー設計用の市販のソフ トウエア、 例えば Ol igoTM [National B i o s c i enc e Inc. (米国） 製]、 GENETYX [ソフ トウエア開発 （株） （日 本） 製] 等を用いることもできる。 Invention (5) is a primer set for PCR amplification of the polynucleotide of invention (3), which is designed based on a known nucleotide sequence, and It can be prepared through each step of synthesis and purification. The following points can be pointed out as points to keep in mind when designing primers. The size (number of bases) of the primer is 15-40 bases, preferably 15-30 bases, in view of satisfying specific annealing with type I DNA. However, when performing LA (long accurate) PCR, at least 30 bases are effective. Avoid the complementary sequences between the primers so that one or two pairs of primers consisting of the sense strand (5, terminal) and the antisense strand (3, terminal) do not anneal to each other. Self-trapping arrangements should also be avoided to prevent the formation of hairpin structures within. Furthermore, to ensure stable binding to type I DNA, the GC content should be about 50%, so that GC-rich or AT-rich is not unevenly distributed in the primer. Since the annealing temperature depends on the melting temperature (Tm), in order to obtain a highly specific PCR product, primers having a Tm value of 55-65 ° C and close to each other are selected. It is also necessary to take care to adjust the final concentration of the primer used in PCR to be about 0.1 to about 1 μ 等. In addition, commercially available software for primer design, for example, Oligo ™ [manufactured by National Bioscience Inc. (USA)], GENETYX [manufactured by Software Development Co., Ltd. (Japan)] and the like can also be used.
発明（6)の抗体はポリ クローナル抗体またはモノクローナル抗体であ り、 発明（1)の抗原ペプチドのェピトープに結合することができる全体 分子、 および Fab、 F (ab' ) 2、 Fv断片等が全て含まれる。 また、 発明（7) の抗体は、 前記発明（6)の抗体とは異なるェピトープに結合する抗体で ある。 このような抗体は、 例えばポリクローナル抗体の場合には、 抗原 ぺプチドやその一部断片を免疫原として動物を免役した後、 血清から得 ることができる。 あるいは、 上記の真核細胞用発現ベクターを注射や遺 伝子銃によって、 動物の筋肉や皮膚に導入した後、 血清を採取すること によって作製することができる。 動物としては、 マウス、 ラッ ト、 ゥサ ギ、 ャギ、 ニヮ トリなどが用いられる。 The antibody of the invention (6) is a polyclonal antibody or a monoclonal antibody, and the whole molecule capable of binding to the epitope of the antigenic peptide of the invention (1), Fab, F (ab ') 2 , Fv fragment and the like are all included. included. The antibody of the invention (7) is an antibody that binds to a different epitope from the antibody of the invention (6). For example, in the case of a polyclonal antibody, such an antibody can be obtained from serum after immunizing an animal using the antigen peptide or a fragment thereof as an immunogen. Alternatively, serum is collected after introducing the above eukaryotic cell expression vector into the muscle or skin of an animal by injection or gene gun. Can be produced by As animals, mice, rats, egrets, goats, and chickens are used.
また、 モノ ク ローナル抗体は、 公知のモノ ク ローナル抗体作製法 (「単クローン抗体」、 長宗香明、 寺田弘共著、 廣川書店、 1990 年； "M onoclonal Antibody James W. Goding, third edition, Academic Pre ss, 1996) に従い作製することができる。  Monoclonal antibodies can be prepared using known monoclonal antibody preparation methods (“Monoclonal Antibodies”, written by Kamei Nagamune and Hiroshi Terada, Hirokawa Shoten, 1990; “Monoclonal Antibody James W. Goding, third edition, Academic Press, 1996).
発明（7)の抗体は、 前記発明（6)の抗体とは異なるェピトープに結合す る抗体である。 このような抗体は、 前記発明（6)の抗体作製のための抗 原べプチド断片とは異なる断片を免疫原とすることによって、 前記と同 様のポリクローナル抗体またはモノクローナル抗体として作製される。 発明（6)および（7)の抗体には、 それぞれ標識物質によって標識化され た抗体も含まれる。 標識物質は、 酵素、 放射性同位体または蛍光色素を 使用することができる。 酵素は、 turnover number が大であること、 抗 体と結合させても安定であること、 基質を特異的に着色させる等の条件 を満たすものであれば特段の制限はなく、 通常のィムノアツセィに用い られる酵素、 例えば、 ペルォキシダーゼ、 一ガラク トシダーゼ、 アル カリフォスファターゼ、 グノレコースォキシダーゼ、 アセチルコリ ンエス テラーゼ、 グルコース一 6—リン酸化脱水素酵素、 リンゴ酸脱水素酵素、 ルシフヱラーゼ等を用いることもできる。 また、 酵素阻害物質や捕酵素 等を用いたり、 アタリジニゥム 'エステルによって標識することもでき る。 これら酵素と抗体との結合は、 マレイミ ド化合物等の架橋剤を用い る公知の方法によって行うことができる。 基質としては、 使用する酵素 の種類に応じて公知の物質を使用することができる。 例えば酵素として ペルォキシダーゼを使用する場合には、 3， 3' , 5， 5'—テ トラメチルベン ジシンを、 また酵素としてアルカリフォスファターゼを用いる場合には、 パラニトロフエノ一ル等を用いることができる。 放射性同位体としては、 125 Iや 3H等の通常の RIA で用いられているものを使用することができ る。 蛍光色素としては、 フルォレツセンスイソチオシァネート （FITC) ゃテトラメチルローダミンイソチオシァネート （TRITC) 等の通常の蛍 光抗体法に用いられるものを使用することができる。 The antibody of the invention (7) is an antibody that binds to a different epitope from the antibody of the invention (6). Such an antibody is produced as a polyclonal antibody or a monoclonal antibody as described above by using, as an immunogen, a fragment different from the antigen peptide fragment for producing the antibody of the invention (6). The antibodies of the inventions (6) and (7) each include an antibody labeled with a labeling substance. As a labeling substance, an enzyme, a radioisotope or a fluorescent dye can be used. There are no particular restrictions on the enzyme as long as it satisfies conditions such as a large turnover number, stability even when bound to the antibody, and specific coloring of the substrate. For example, peroxidase, monogalactosidase, alkaline phosphatase, gnorecosoxidase, acetylcholinesterase, glucose-16-phosphoryl dehydrogenase, malate dehydrogenase, luciferase and the like can be used. In addition, it is also possible to use an enzyme inhibitor, a capture enzyme, or the like, or label with ataridinium'ester. The binding between the enzyme and the antibody can be performed by a known method using a crosslinking agent such as a maleimide compound. As the substrate, a known substance can be used depending on the type of the enzyme to be used. For example, when peroxidase is used as the enzyme, 3,3 ', 5,5'-tetramethylbenzicine can be used. When alkaline phosphatase is used as the enzyme, paranitrophenol can be used. As radioisotopes, Those used in normal RIA such as 125 I and 3 H can be used. As the fluorescent dye, those used in a usual fluorescent antibody method, such as fluorescein isothiocyanate (FITC) ゃ tetramethylrhodamine isothiocyanate (TRITC), can be used.
発明（8)のヒ ト血管炎診断方法は、 被験者から採取した生体試料中の、 前記発明（1)の抗原べプチドと結合する抗体を検出し、 生体試料中にそ の抗体が健常者よりも多く存在する被験者を血管炎患者または血管炎ハ ィ リスク者と判定する。 すなわち、 発明（1)の抗原ペプチドは、 血管炎 患者の生体試料中の抗体と結合するぺプチドであるから、 被験者の生体 試料と反応させ、 この抗原ペプチドと結合する抗体を健常者よりも多く 含む生体試料を、 血管炎患者またはそのハイリスク患者の生体試料とし て判定することができる。 抗体の検出は、 RIA、 ELISA、 EIA、 CLIA、 CLE IA、 ルシフェラーゼアツセィ等の各種公知ィムノアッセィとして実施す ることができる。 本発明における検出は、 定性的であっても定量的であ つてもよく、 抗体を定量する場合は、 血管炎の診断、 スク リーニング目 的のほか、 血管炎治療のモニタリ ング目的としても使用できる。 生体試 料としては、 血液、 血清、 血漿や血液細胞 （単核球等）、 尿、 骨髄液、 唾液等を用いることができる。 なおその際に、 すでに知られている他の 血管炎マーカーを併用することもできる。  The method for diagnosing human vasculitis according to the invention (8) comprises detecting an antibody that binds to the antigenic peptide of the invention (1) in a biological sample collected from a subject, and detecting the antibody in the biological sample from a healthy subject. Subjects with high levels of vasculitis are determined to be vasculitis patients or high-risk vasculitis patients. That is, since the antigenic peptide of the invention (1) is a peptide that binds to an antibody in a biological sample of a vasculitis patient, the antigenic peptide is reacted with a biological sample of a subject, and more antibodies bind to this antigenic peptide than in a healthy subject. Can be determined as a biological sample of a vasculitis patient or a high-risk patient thereof. Detection of the antibody can be carried out as various known immunoassays such as RIA, ELISA, EIA, CLIA, CLEIA, and luciferase atsey. The detection in the present invention may be either qualitative or quantitative. When the antibody is quantified, it can be used for the purpose of diagnosing and screening vasculitis and also for monitoring the treatment of vasculitis. . As a biological sample, blood, serum, plasma, blood cells (eg, mononuclear cells), urine, bone marrow fluid, saliva, and the like can be used. At this time, other known vasculitis markers can also be used in combination.
具体的な診断は、 例えば抗原ペプチドに被験者血清を接触させ、 抗原 ぺプチドと被験者血清中の IgG抗体とを液相中において反応させる。 さ らに血清中の IgG抗体と特異的に結合する標識化抗ヒ ト IgG抗体を反応 させて、 標識化抗ヒ ト IgG抗体のシグナルを検出すればよい。 標識化抗 ヒ ト IgG抗体の標識物質は、 前記の標識化抗体において例示したような 酵素、 放射性同位体または蛍光色素等を使用することができる。 酵素を 用いる場合には、 酵素作用によって分解して発色する基質を加え、 基質 の分解量を光学的に測定することによって酵素活性を求め、 これを結合 抗体量に換算し、 標準値との比較から抗体量が算出される。 放射性同位 体を用いる場合には、 放射性同位体の発する放射線量をシンチレーショ ンカウンタ一等により測定する。 また、 蛍光色素を用いる場合には、 蛍 光顕微鏡を組み合わせた測定装置によって蛍光量を測定すればよい。 化 学発光法の場合はァクリジニゥム . エステル発光の量を測定すればよい c シグナルの検出は、 例えば、 ウェスタンプロッ ト分析を採用すること ができる。 あるいは、 抗原ペプチド +生体試料中抗体 +標識化抗ヒ ト I gG 抗体の結合体を、 公知の分離手段 （クロマト法、 固相法等） によつ て分離し、 標識化抗ヒ ト IgG抗体のシグナルを検出するようにしてもよ い。 なお、 このような診断方法の簡便かつ広範囲な実施を可能とするも のと して、 発明（13)の診断キッ トが提供される。 For specific diagnosis, for example, the serum of the subject is brought into contact with the antigen peptide, and the antigen peptide is reacted with the IgG antibody in the serum of the subject in a liquid phase. Furthermore, a signal of the labeled anti-human IgG antibody may be detected by reacting with a labeled anti-human IgG antibody that specifically binds to the IgG antibody in serum. As a labeling substance of the labeled anti-human IgG antibody, an enzyme, a radioisotope, a fluorescent dye or the like as exemplified in the above-mentioned labeled antibody can be used. When using an enzyme, add a substrate that decomposes and develops color by the action of the enzyme. The enzyme activity is determined by optically measuring the amount of degradation of the enzyme, and this is converted into the amount of bound antibody. When using a radioisotope, measure the radiation dose emitted by the radioisotope using a scintillation counter or the like. When a fluorescent dye is used, the amount of fluorescence may be measured by a measuring device combined with a fluorescence microscope. In the case of the chemical luminescence method, it is sufficient to measure the amount of acridinium.ester luminescence. For the detection of the c signal, for example, Western plot analysis can be employed. Alternatively, the conjugate of the antigen peptide + the antibody in the biological sample + the labeled anti-human IgG antibody is separated by a known separation method (chromatography, solid phase method, etc.), and the labeled anti-human IgG antibody is separated. Signal may be detected. In addition, the diagnostic kit of the invention (13) is provided to enable such a diagnostic method to be performed simply and widely.
発明（8)の診断方法はまた、 抗原べプチドの 1種類以上を担体に固定 化し、 この担体上において被験者生体試料の担体との結合を試験する方 法 （発明（9) ) として実施することもできる。 抗原ペプチドを担体上に 固定化することによって、 未結合の標識化結合分子を容易に除去するこ とができる。 担体としては、 ビーズ、 容器内壁、 微粒子、 多孔質担体、 あるいは磁性粒子などが用いられる。 これらの固相は、 ポリスチレン、 ポリカーボネート、 ポリ ビニルトルエン、 ポリプロピレン、 ポリエチレ ン、 ポリ塩化ビュル、 ナイロン、 ポリメタクリ レート、 ラテックス、 ゼ ラチン、 ァガロース、 ガラス、 金属、 あるいはセラミ ック等の素材を利 用して成型されたものを利用できる。 これらの固相素材の表面に、 抗体 等を化学的に結合するための官能基を導入した固相素材も知られている。 固相と抗体 （あるいは抗原） についても、 ポリ - L-リジンゃグルタール アルデヒ ド処理といった化学的な結合や、 物理吸着といった公知の結合 方法を応用することができる。 また、 このような診断方法の簡便かつ広 範囲な実施を可能とするものとして、 発明（14)の診断キッ トが提供され る。 また特に、 数十種類の抗原ペプチドを固定化したメンブレンを用い るプロテインアレイ法では、 0. 01ml 程度の被験者血清を用いて多種類 の抗体の発現を短時間で解析することができる。 The diagnostic method of the invention (8) may be carried out as a method (invention (9)) in which one or more antigen peptides are immobilized on a carrier, and the binding of the subject biological sample to the carrier is tested on the carrier. You can also. By immobilizing the antigen peptide on the carrier, unbound labeled binding molecules can be easily removed. As the carrier, beads, container inner walls, fine particles, porous carriers, magnetic particles, or the like are used. These solid phases utilize materials such as polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, latex, gelatin, agarose, glass, metal, or ceramic. Can be used. Solid phase materials in which a functional group for chemically binding an antibody or the like is introduced to the surface of these solid phase materials are also known. For the solid phase and the antibody (or antigen), well-known binding methods such as chemical binding such as poly-L-lysine-glutalaldehyde treatment and physical adsorption can be applied. In addition, such a diagnostic method is simple and broad. The diagnostic kit of the invention (14) is provided to enable a wide range of implementation. In particular, in the protein array method using a membrane on which several tens of antigen peptides are immobilized, the expression of various antibodies can be analyzed in a short time using about 0.01 ml of the serum of the subject.
この出願の発明（10)の診断方法は、 被験者の生体試料中の、 前記発明 (6)の抗体、 またはその標識化抗体と結合する抗原べプチドを検出し、 試料中にその抗原べプチドが健常者よりも多く存在する被験者を血管炎 患者またはそのハイリスク者と判定する。 すなわち、 ここで使用する抗 体または標識化抗体は、 血管炎を呈した血管内皮細胞で発現している抗 原ペプチドと特異的に結合する抗体であるから、 この抗体と結合する抗 原ぺプチドを含む生体試料を、 血管炎患者またはそのハイリスク患者の 試料として判定することができる。 生体試料としては、 血液、 血清、 血 漿、 血液細胞 （単核球等）、 尿、 骨髄液、 唾液等を対象とすることがで きる。  The diagnostic method according to the invention (10) of the present application comprises detecting an antigen peptide that binds to the antibody of the invention (6) or a labeled antibody thereof in a biological sample of a subject, and the antigen peptide is detected in the sample. Subjects present more than healthy subjects are determined to be vasculitis patients or their high-risk subjects. That is, since the antibody or labeled antibody used here is an antibody that specifically binds to an antigen peptide expressed in vascular endothelial cells exhibiting vasculitis, the antigen peptide that binds to this antibody is used. Can be determined as a sample of a vasculitis patient or a high-risk patient thereof. Biological samples can include blood, serum, plasma, blood cells (eg, mononuclear cells), urine, bone marrow fluid, saliva, and the like.
この発明（10)の診断方法における一つの態様は、 抗体と抗原ペプチド との結合を液相系において行う方法である。 例えば、 発明（6)の標識化 抗体と生体試料とを接触させて標識化抗体と抗原ぺプチドを結合させ、 この結合体を前記発明（8)と同様の方法で分離し、 標識シグナルを同様 の方法で検出する。 このような診断方法の簡便かつ広範囲な実施を可能 とするものとして、 発明（15)の診断キッ トが提供される。  One embodiment of the diagnostic method of the present invention (10) is a method in which the antibody is bound to the antigen peptide in a liquid phase system. For example, the labeled antibody of the invention (6) is brought into contact with a biological sample to bind the labeled antibody and the antigen peptide, the conjugate is separated by the same method as in the invention (8), and the labeling signal is similarly determined. Method. The diagnostic kit according to the invention (15) is provided as a method that enables such a diagnostic method to be carried out simply and widely.
液相系での診断の別の方法は、 発明（6)の抗体 （一次抗体） と生体試 料とを接触させて一次抗体と抗原べプチドを結合させ、 この結合体に標 識化した発明（7)の抗体 （二次抗体） を結合させ、 この三者の結合体に おける標識シグナルを検出する。 あるいは、 さらにシグナルを増強させ るためには、 非標識の二次抗体を先ず抗体 +抗原ペプチド結合体に結合 させ、 この二次抗体に標識物質を結合させるようにしてもよい。 このよ うな二次抗体への標識物質の結合は、 例えば二次抗体をビォチン化し、 標識物質をアビジン化しておくことによって行うことができる。 あるい は、 二次抗体の一部領域 （例えば、 Fc 領域） を認識する抗体 （三次抗 体） を標識し、 この三次抗体を二次抗体に結合させるようにしてもよい c なお、 一次抗体と二次抗体は、 両方ともモノクローナル抗体を用いるこ ともでき、 あるいは、 一次抗体と二次抗体のいずれか一方をポリクロー ナル抗体とすることもできる。 液相からの結合体の分離やシグナルの検 出は前記発明（8)と同様とすることができる。 また、 このような診断方 法の簡便かつ広範囲な実施を可能とするものとして、 発明（16)の診断キ ッ トが提供される。 Another method of diagnosis in a liquid phase system is the method in which the antibody (primary antibody) of the invention (6) is brought into contact with a biological sample to bind the primary antibody and the antigen peptide, and the invention is labeled with this conjugate. The antibody (secondary antibody) of (7) is allowed to bind, and the labeling signal of the conjugate is detected. Alternatively, in order to further enhance the signal, an unlabeled secondary antibody may first be bound to the antibody + antigen peptide conjugate, and a labeling substance may be bound to this secondary antibody. This Such binding of the labeling substance to the secondary antibody can be performed, for example, by biotinylation of the secondary antibody and avidinization of the labeling substance. Or, part of the region of the secondary antibody (eg, Fc region) and labeled antibody (tertiary antibody) recognizes, good c Note also the tertiary antibody so as to bind to the second antibody, primary antibody For both the secondary antibody and the secondary antibody, a monoclonal antibody can be used, or one of the primary antibody and the secondary antibody can be a polyclonal antibody. Separation of the conjugate from the liquid phase and detection of the signal can be performed in the same manner as in the invention (8). In addition, the diagnostic kit of the invention (16) is provided as a method which enables simple and wide-ranging implementation of such a diagnostic method.
発明（10)の診断方法における別の態様は、 抗体と抗原べプチドとの結 合を固相系において試験する方法である。 この固相系における方法は、 極微量の抗原べプチド検出と操作の簡便化のため好ましい方法である。 すなわちこの固相系の方法は、 発明（6)の抗体を樹脂プレート、 ビーズ- メンブレン等の担体に固定化し、 この固定化抗体に抗原ペプチドを結合 させ、 非結合ペプチドを洗浄除去した後、 担体上に残った抗体 +抗原べ プチド結合体に発明（7)の標識化抗体を結合させて、 この標識化抗体の シグナルを検出する方法である。 この方法は、 いわゆる 「サンドイッチ 法」 と呼ばれる方法であり、 マーカーとして酵素を用いる場合には、 「E LISA enzyme l inked immunosorbent assayノ」 と して広 用レヽられてレヽ る方法である。 2 種類の抗体は、 両方ともモノクローナル抗体を用いる こともでき、 あるいは、 いずれか一方をポリクローナル抗体とすること もできる。 シグナルの検出は前記発明（8)と同様とすることができる。 また、 このような診断方法の簡便かつ広範囲な実施を可能とするものと して、 発明（17)の診断キッ トが提供される。  Another embodiment of the diagnostic method of the invention (10) is a method of testing the binding between an antibody and an antigen peptide in a solid phase system. This method using a solid phase system is a preferred method for detecting a trace amount of antigen peptide and simplifying the operation. That is, this solid-phase method involves immobilizing the antibody of the invention (6) on a carrier such as a resin plate or a bead-membrane, binding the antigen peptide to the immobilized antibody, washing away the unbound peptide, and then removing the carrier. In this method, the labeled antibody of the invention (7) is bound to the antibody + antigen peptide conjugate remaining above, and the signal of the labeled antibody is detected. This method is a so-called “sandwich method”. When an enzyme is used as a marker, the method is widely used as “ELISA enzyme linked immunosorbent assay”. The two types of antibodies can both be monoclonal antibodies, or one of them can be a polyclonal antibody. Detection of the signal can be performed in the same manner as in the invention (8). Further, the diagnostic kit of the invention (17) is provided so as to enable simple and wide-ranging implementation of such a diagnostic method.
発明（13)〜（17)の診断キッ トは、 前記発明（8)〜（1 1)の診断方法を行 うための試薬キッ トである。 このようなキッ トは、 被検成分の種類に応 じて各種のものが市販されており、 この発明の診断キッ トも、 この発明 によって提供される抗原べプチド、 抗体およびノまたは標識化抗体を用 いることを除き、 公知公用のキッ トに用いられている各要素によって構 成することができる。 The diagnostic kits of Inventions (13) to (17) implement the diagnostic methods of Inventions (8) to (11). It is a reagent kit for use. Various types of such kits are commercially available depending on the type of the test component. The diagnostic kits of the present invention also include the antigen peptides, antibodies, and labeled antibodies provided by the present invention. Except for the use of a publicly-available kit, it can be composed of the components used in a publicly-available kit.
発明（12)の診断方法は、 被験者の生体試料における前記発明（3)のポ リヌクレオチドの存在量を試験し、 ポリヌクレオチドの存在量が健常者 のそれらと比較して多い被験者を血管炎患者またはそのハイリスク者と 判定する。 具体的な判定基準としては、' 被験者のポリヌクレオチドの存 在量が健常者のそれと比較して、 10%以上、 好ましくは 30°/。以上、 さら に好ましくは 70%以上、 最も好ましくは 100%以上である場合である。 生体試料としては、 便や血液、 血液細胞 （リ ンパ球等） を対象とする ことができる。 ポリヌクレオチドの検出、 測定は公知の PCR法や RT- PC R法、 定量的 RT- PCR法等によって行うことができ、 その場合の PCR は 発明（5)のプライマーセッ トを用いることができる。  The diagnostic method of the invention (12) is a method for testing the amount of the polynucleotide of the invention (3) in a biological sample of a subject, and determining whether the subject has a greater amount of the polynucleotide than that of a healthy subject. Or, it is judged as a high risk person. As a specific criterion, the abundance of the polynucleotide of the subject is 10% or more, preferably 30 ° /, as compared with that of the healthy subject. As mentioned above, it is more preferably 70% or more, most preferably 100% or more. The biological sample may be stool, blood, or blood cells (such as lymphocytes). Polynucleotide detection and measurement can be performed by a known PCR method, RT-PCR method, quantitative RT-PCR method, or the like. In that case, the primer set of the invention (5) can be used for PCR.
またさらに、 発明（12)の診断方法は、 この発明によって提供されるポ リヌクレオチドまたはオリゴヌクレオチドを備えた DNAマイクロアレイ によっても実施することができる。 マイクロアレイの作製方法としては、 固相担体表面で直接オリゴヌクレオチドを合成する方法 （オン · チップ 法） と、 予め調製したオリゴヌクレオチドを固相担体表面に固定する方 法とが知られている。 この発明で使用するマイクロアレイは、 このいず れの方法でも作製することができる。 オン · チップ法としては、 光照射 で選択的に除去される保護基の使用と、 半導体製造に利用されるフォ ト リソグラフィ 技術および固相合成技術とを組み合わせて、 微少なマト リ ックスの所定の領域での選択的合成を行う方法 （マスキング技術：例 えば、 Fodor, S. P. A. Sc ience 251 : 767， 1991 ) 等によって行うことが できる。 一方、 予め調製したオリゴヌクレオチドを固相担体表面に固定 する場合には、 官能基を導入したオリゴヌク レオチ ドを合成し、 表面処 理した固相担体表面にオリ ゴヌクレオチドを点着し、 共有結合させる (例えば、 Lamture, J. B. et al. Nucl. Acids Res. 22: 212ト 2125， 19 94； Guo, Z. et al. Nucl. Acids Res. 22:5456-5465， 1994)。 オリ ゴ ヌク レオチ ドは、 一般的には、 表面処理した固相担体にスぺーサーゃク ロスリンカーを介して共有結合させる。 ガラス表面にポリアクリルァミ ドゲルの微小片を整列させ、 そこに合成ォリゴヌクレオチドを共有結合 させる方法も知られている (Yershov, G. et al. Proc. Natl. Acad. S ci. USA 94:4913， 1996)。 また、 シリカマイクロアレイ上に微小電極の ァレイを作製し、 電極上にはストレプトァビジンを含むァガロースの浸 透層を設けて反応部位とし、 この部位をプラスに荷電させることでピオ チン化オリゴヌクレオチドを固定し、 部位の荷電を制御することで、 高 速で厳密なハイブリダィゼーシヨ ンを可能にする方法も知られている (Sosnowski, R. G. et al. Proc. Natl. Acad. Sci. USA 94: 1119 - 1123, 1997)。 このマイクロアレイを使用して血管炎を診断する場合には、 例 えば被験者の細胞から単離した mRNA を铸型として、 cDNAを合成し、 PC R増幅する。 その際に、 標識 dNTPを取り込ませて標識 cDNA とする。 こ の標識 cDNA をマクロアレイに接触させ、 マイクロアレイのキヤプチャ —プローブ （オリゴヌクレオチドまたはポリヌクレオチド） にハイブリ ダイズした cDNAを検出する。 ハイプリダイゼーシヨ ンは、 96穴もしく は 384穴プラスチックプレートに分注して標識 cDNA水性液を、 マイク 口ァレイ上に点着することによって実施することができる。 点着の量は、 l〜100nl 程度とすることができる。 ハイブリダィゼーシヨ ンは、 室温 〜70°Cの温度範囲で、 6〜20 時間の範囲で実施することが好ましい。 ハ ィプリダイゼーシヨ ン終了後、 界面活性剤と緩衝液との混合溶液を用い て洗浄を行い、 未反応の標識 cDNA を除去する。 界面活性剤としては、 ドデシル硫酸ナト リ ウム （SDS) を用いることが好ましい。 緩衝液とし ては、 クェン酸緩衝液、 リ ン酸緩衝液、 ホウ酸緩衝液、 トリス緩衝液、 グッ ド緩衝液等を用いることができるが、 クェン酸緩衝液を用いること が好ましい。 Still further, the diagnostic method of the invention (12) can be carried out by a DNA microarray provided with the polynucleotide or the oligonucleotide provided by the present invention. As a method for preparing a microarray, a method of directly synthesizing an oligonucleotide on the surface of a solid support (on-chip method) and a method of immobilizing a previously prepared oligonucleotide on the surface of the solid support are known. The microarray used in the present invention can be produced by any of these methods. The on-chip method combines the use of protective groups that are selectively removed by light irradiation with the photolithography technology and solid-phase synthesis technology used in semiconductor manufacturing, to achieve the specified fine-grained matrix. Selective synthesis in a region (masking technique: for example, Fodor, SPA Science 251: 767, 1991) it can. On the other hand, when immobilizing a previously prepared oligonucleotide on the surface of the solid support, the oligonucleotide with the functional group introduced is synthesized, and the oligonucleotide is spotted on the surface of the surface-treated solid support to form a covalent bond. (E.g., Lamture, JB et al. Nucl. Acids Res. 22: 212 2125, 1994; Guo, Z. et al. Nucl. Acids Res. 22: 5456-5465, 1994). Oligonucleotides are generally covalently bonded to a surface-treated solid support via spacer crosslinkers. A method is also known in which polyacrylamide gel micro-pieces are aligned on a glass surface and a synthetic oligonucleotide is covalently bonded thereto (Yershov, G. et al. Proc. Natl. Acad. Sci. USA 94: 4913, 1996). In addition, an array of microelectrodes was prepared on a silica microarray, an agarose infiltration layer containing streptavidin was provided on the electrode to serve as a reaction site, and this site was positively charged to allow the biotinylated oligonucleotide to be charged. It is also known to enable fast and strict hybridization by fixing and controlling the charge of the site (Sosnowski, RG et al. Proc. Natl. Acad. Sci. USA 94 : 1119-1123, 1997). When diagnosing vasculitis using this microarray, cDNA is synthesized and PCR amplified, for example, by using mRNA isolated from the cells of the subject as type III. At this time, the labeled dNTPs are incorporated into a labeled cDNA. The labeled cDNA is brought into contact with the macroarray to detect the cDNA hybridized to the microarray capture probe (oligonucleotide or polynucleotide). Hybridization can be carried out by dispensing a 96-well or 384-well plastic plate and applying an aqueous solution of the labeled cDNA on a microphone array. The amount of spotting can be on the order of l-100 nl. The hybridization is preferably performed at a temperature in the range of room temperature to 70 ° C. for 6 to 20 hours. After the hybridization, use a mixed solution of surfactant and buffer. Wash to remove unreacted labeled cDNA. It is preferable to use sodium dodecyl sulfate (SDS) as the surfactant. As the buffer, a citrate buffer, a phosphate buffer, a borate buffer, a Tris buffer, a good buffer, and the like can be used, but a citrate buffer is preferably used.
発明（19)の血管炎の治療または予防方法は、 患者から血液を取り出し て血液中の抗ヒ トペルォキシレドキシン抗体を選択的に除去した後、 血 液を再び患者に戻す方法である。 発明（19)の方法は、 「（a)患者から採取 した血液または抗体を含むその分画を、 血液に不溶性の担体に固定化さ れたヒ トのペルォキシレドキシンまたはその抗原決定基を含む断片に接 触させる工程」 （以下、 「接触工程」 と略称する。） および 「（b)工程（a) の後に当該血液またはその分画を、 患者に戻す工程」 を含む。 実施例に 後述するように、 本発明者らは血管炎患者の血清中には抗ヒ トペルォキ シレドキシン抗体が有意に高く存在することを初めて確認した。 この結 果からは、 抗ヒ トペルォキシレドキシン抗体が血管炎の発症または進展 に強く関連することが示唆される。 したがって、 血液中の抗ヒ トペルォ キシレドキシン抗体を除去することにより血管炎の症状の改善または予 防が可能になると考えられる。 発明（19)の方法では、 接触工程において. 固定化されたヒ トのペルォキシレドキシンまたはその抗原決定基を含む 断片によって抗ヒ トペルォキシレドキシン抗体が吸着されることによつ て、 上記抗体が血液中から除去される。  The method for treating or preventing vasculitis according to the invention (19) is a method of removing blood from a patient, selectively removing anti-human peroxyredoxin antibody in the blood, and then returning the blood to the patient again. The method according to the invention (19) is characterized in that “(a) a blood peroxyredoxin or an antigenic determinant thereof obtained by immobilizing blood or a fraction thereof containing antibodies collected from a patient on a carrier insoluble in blood. (Hereinafter referred to as “contact step”) and “(b) returning the blood or a fraction thereof to the patient after step (a)”. As described later in the Examples, the present inventors have confirmed for the first time that the serum of a patient with vasculitis has a significantly high anti-hyperoxyredoxin antibody. The results suggest that anti-hyperperoxyredoxin antibodies are strongly associated with the development or progression of vasculitis. Therefore, it is considered that the elimination of anti-hyperoxyredoxin antibody in blood can improve or prevent the symptoms of vasculitis. In the method of the invention (19), the anti-human peroxyredoxin antibody is adsorbed by the immobilized human peroxyredoxin or a fragment containing the antigenic determinant thereof in the contacting step. The antibodies are removed from the blood.
患者から取り出した血液は、 ヒ トペルォキシレドキシン等と接触させ る前に、 いくつかの分画に分ける処理を施してもよい。 例えば発明（20) のように、 血液を血漿分離機で血漿分画と細胞分画に分離し、 抗ヒ トぺ ルォキシレドキシン抗体が存在する血漿分画のみを上記接触工程に付す ことができる。 抗ヒ トペルォキシレドキシン抗体が除去された血漿は、 細胞分画と共に再び患者の体内へ戻される。 Blood taken from a patient may be subjected to treatment to separate it into several fractions before contacting with human peroxyredoxin or the like. For example, as in the invention (20), blood is separated into a plasma fraction and a cell fraction by a plasma separator, and only the plasma fraction in which an anti-hydroxy redoxin antibody is present is subjected to the contacting step. it can. Plasma from which anti-human peroxyredoxin antibodies have been removed It is returned to the patient's body together with the cell fraction.
発明（19)の方法を実施するには、 発明（18)の治療剤を用いることがで きる。 発明（18)の治療剤は、 ヒ トペルォキシレドキシンまたはその抗原 決定基を含んだ断片が血液に不溶性の担体に固定化された状態で含まれ ている。 したがって、 患者から取り出された血液を該治療剤に通過させ ると、 血液中の抗ヒ トペルォキシレドキシン抗体を選択的に除去するこ とが可能である。 血液に不溶性の担体として、 セルロース、 ァガロース, セファロース、 デキス トラン、 キチン、 キトサン、 これらの誘導体、 有 機または無機の多孔性材料、 磁気ビーズ、 マイクロビーズなどを使用す ることができるが、 血液に不溶性である限りこれらに限定されない。 上 記担体にヒ トペルォキシレドキシンまたはその抗原決定基を含んだ断片 を物理的吸着や共有結合による不溶化によって固定化させ、 発明（18)の 治療剤とすることができる。  In order to carry out the method of the invention (19), the therapeutic agent of the invention (18) can be used. The therapeutic agent of the invention (18) contains human peroxyredoxin or a fragment containing the antigenic determinant thereof in a state immobilized on a blood-insoluble carrier. Therefore, when blood extracted from a patient is passed through the therapeutic agent, it is possible to selectively remove the anti-hyperperoxyredoxin antibody in the blood. As a carrier insoluble in blood, cellulose, agarose, sepharose, dextran, chitin, chitosan, derivatives thereof, organic or inorganic porous materials, magnetic beads, microbeads, etc. can be used. It is not limited to these as long as they are insoluble. The therapeutic agent of the invention (18) can be obtained by immobilizing a fragment containing a hyperperoxyredoxin or an antigenic determinant thereof on the carrier by physical adsorption or insolubilization by covalent bond.
なお本明細書において引用された全ての先行技術文献は、 参照として 本明細書に組み入れられる。 図面の簡単な説明  All prior art documents cited in the present specification are incorporated herein by reference. BRIEF DESCRIPTION OF THE FIGURES
図 l a は、 細胞抽出タンパク質を 2次元電気泳動法で分離展開し、 ク マシー染色にて可視化した写真である。 図 l b は、 展開したタンパク質 をニトロセルロース膜に転写し、 血管炎患者 4名の血清と反応させた写 真である。  Figure la is a photograph of a cell extracted protein separated and developed by two-dimensional electrophoresis and visualized by Coomassie staining. Figure lb is a photograph of the developed protein transferred to a nitrocellulose membrane and reacted with the sera of four patients with vasculitis.
図 2は、 MBPおよび融合タンパク質 MBP- BC452 を 1次元電気泳動し、 ペルォキシダーゼ標識 Ni- NTAで染色した結果を示す写真である。  FIG. 2 is a photograph showing the results of one-dimensional electrophoresis of MBP and the fusion protein MBP-BC452 and staining with peroxidase-labeled Ni-NTA.
図 3は、 組換え BC000452 を抗原ペプチドとして固相化した ELISA に よる血管炎診断の結果を示す写真である。 発明を実施するための最良の形態 FIG. 3 is a photograph showing the results of a diagnosis of vasculitis by ELISA in which recombinant BC000452 was immobilized as an antigen peptide. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 実施例を示してこの出願の発明についてさらに詳細かつ具体的 に説明するが、 この出願の発明は以下の例によって限定されるものでは ない。  Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention of this application is not limited to the following examples.
実施例 1 ： 血管炎抗原ペプチドの同定 Example 1: Identification of vasculitis antigen peptide
ヒ ト臍帯静脈由来內皮細胞 （HUVEC) と HeLa細胞 （対照） を用い、 そ れぞれから尿素、 チォ尿素、 CHAPS を含む溶液でタンパク質を溶出し、 これを等電点電気泳動おょぴ分子量による SDS- PAGE を組み合わせた 2 次元電気泳動法で分離展開し、 クマシ一染色にて可視化した （図 la)。 次いで、 展開したタンパク質をさらにニ トロセルロース膜に転写し、 血 管炎患者 4名の血清と反応させた （図 lb)。 患者血清中抗体と反応する タンパク質 （自己抗原ペプチド） が、 図 lb 中に番号で示したものを含 め、 複数個同定された。 .  Using human umbilical vein-derived skin cells (HUVEC) and HeLa cells (control), proteins were eluted from each with a solution containing urea, thiourea, and CHAPS, which were then subjected to isoelectric focusing electrophoresis. Separated and developed by two-dimensional electrophoresis combined with SDS-PAGE, and visualized with Kumashi-stain (Figure la). Next, the developed protein was further transferred to a nitrocellulose membrane and reacted with the sera of four patients with vasculitis (Fig. Lb). Several proteins (autoantigen peptides) that reacted with antibodies in patient sera were identified, including those indicated by numbers in the figure lb. .
次に、 図 laのゲルから、 図 lbで同定された陽性タンパク質スポッ ト 28 と一致するタンパク質を切り出して回収し、 トリプシン消化によつ てゲルから抽出した。 この消化べプチドの質量を飛行時間型質量分析器 を用いた mass- f ingerprinting法により決定し、 検索ソフ トウェア （マ スコッ ト） によるタンパク質データベース検索から質量の一致する侯補 タンパク質を選択した。  Next, a protein corresponding to the positive protein spot 28 identified in FIG. Lb was excised and recovered from the gel in FIG. La, and extracted from the gel by trypsin digestion. The mass of this digested peptide was determined by the mass-fingerprinting method using a time-of-flight mass spectrometer, and a candidate protein having a matching mass was selected from a protein database search using search software (mascot).
その結果、 この抗原ペプチドが、 PRDX2 相同体 （GenBank/BC000452) であることが確認された （以下、 このペプチドを 「BC452」 と記載する ことがある）。  As a result, it was confirmed that this antigen peptide was a PRDX2 homolog (GenBank / BC000452) (hereinafter, this peptide may be referred to as “BC452”).
実施例 2 ： BC452の抗原性の確認 Example 2: Confirmation of antigenicity of BC452
実施例 1で同定した BC452が、 真に患者自己抗体に反応する抗原であ るかを確かめるために、 組換え BC452を作成した。  To confirm that the BC452 identified in Example 1 was truly an antigen that reacts with patient autoantibodies, a recombinant BC452 was created.
臍帯静脈由来血管内皮細胞より調整した mRNAから RT- PCR法により、 BC452 cDNA (配列番号 1 ) のコード領域を増幅し、 大腸菌蛋白発現べク ター （pMAL- cHi s) にクローニング後、 大腸菌に導入し、 マルトース結 合タンパク質 （MBP) との融合タンパク質として発現させた。 RT-PCR from mRNA prepared from umbilical vein-derived vascular endothelial cells The coding region of BC452 cDNA (SEQ ID NO: 1) was amplified, cloned into E. coli protein expression vector (pMAL-cHis), introduced into E. coli, and expressed as a fusion protein with maltose binding protein (MBP) .
結果は図 2a に示したとおりである。 この図 2a は、 MBP (コントロー ル） および融合タンパク質 MBP- BC452を精製の後、 1次元電気泳動し、 さらに、 各タンパク質の C端に位置するヒスチジンタグを認識する Ni- NTA で染色した結果であり、 それぞれのタンパク質が産生精製が確認さ れた。  The results are shown in Figure 2a. Figure 2a shows the results of purification of MBP (control) and fusion protein MBP-BC452, followed by one-dimensional electrophoresis and staining with Ni-NTA that recognizes the histidine tag located at the C-terminal of each protein. Yes, production and purification of each protein were confirmed.
次いで、 MBPおよび融合タンパク質 MBP- BC452 を同一レーンで展開し, 個別に各種血管炎患者血清と反応させた。 結果は図 2b に示したとおり であり、 上部のバンド （融合タンパク質 MBP-BC452) 力 S MBP に比較して 格段に濃く染色されたことから、 BC452 に対する自己抗体が各種血管炎 患者血清血清中に存在することが確認された。  Next, MBP and the fusion protein MBP-BC452 were developed in the same lane and reacted individually with sera of various vasculitis patients. The results are shown in Figure 2b. The upper band (fusion protein MBP-BC452) showed a much stronger staining than the S-MBP, indicating that autoantibodies against BC452 were present in the serum of patients with various vasculitis patients. Confirmed to be present.
実施例 3 ： ELISAによる診断 Example 3: Diagnosis by ELISA
実施例 2で調製した組換え BC452を抗原ペプチドとして固相化し、 被 検血清を反応させ、 さらにペルォキシダーゼ標識抗ヒ ト IgG抗体を反応 させて、 ペルォキシダーゼの基質を加えて発色させ、 抗原ペプチドと血 清中抗体との結合を検出した。 被験血清として、 血管炎患者 （ゥェゲナ 一肉芽腫症、 顕微鏡的多発血管炎、 アレルギー性肉芽腫性血管炎、 結節 性多発動脈炎、 側頭動脈炎、 高安病、 悪性関節リ ウマチ、 バージャ一病、 全身性エリテマトーデス）、 血管炎の無い膠原病患者、 健常人の血清を 用いた。  The recombinant BC452 prepared in Example 2 was immobilized as an antigen peptide, reacted with a test serum, and further reacted with a peroxidase-labeled anti-human IgG antibody, and a peroxidase substrate was added to develop a color. Binding to the Seichu antibody was detected. As test serum, patients with vasculitis (Pegena monogranulomatosis, microscopic polyangiitis, allergic granulomatous vasculitis, polyarteritis nodosa, temporal arteritis, Takayasu disease, rheumatoid arthritis, Bajaja disease Sera from systemic lupus erythematosus), patients with collagen disease without vasculitis, and healthy individuals.
図 3 は、 発色反応を 0D値として数値化したものである。 健常人の平 均 0D値にその標準偏差の 3倍を加えた値を 100 単位と定義し、 それよ り単位数の高い血清を陽性と判定した。 BC452 に対する自己抗体は血管 炎患者の 68 %で検出され、 血管炎の無い膠原病患者では 15%、 健常人 では 0%であった。 この結果から、 抗原ペプチド BC452対する自己抗体 の検出は血管炎を診断する上で極めて有用なマーカーとなることが確認 された。 産業上の利用の可能性 Figure 3 is a numerical representation of the color reaction as a 0D value. The value obtained by adding three times the standard deviation to the mean 0D value of a healthy person was defined as 100 units, and sera with a higher number of units were determined to be positive. Autoantibodies to BC452 were detected in 68% of patients with vasculitis, 15% in patients with collagen disease without vasculitis, healthy Was 0%. From these results, it was confirmed that the detection of an autoantibody to the antigen peptide BC452 is a very useful marker for diagnosing vasculitis. Industrial potential
以上詳しく説明したとおり、 この出願の発明によって、 血管炎の診断 用マーカーとして有用な新規抗原べプチドと、 これらを用いた血管炎診 断方法が提供される。 これによつて、 血管炎の早期かつ高精度の診断が 可能となる。  As described above in detail, the invention of this application provides a novel antigenic peptide useful as a diagnostic marker for vasculitis and a method for diagnosing vasculitis using the same. This enables early and highly accurate diagnosis of vasculitis.

Claims

請求の範囲 ヒ ト · ペルォキシレドキシンである血管炎抗原べプチド。 Claims A vasculitis antigen peptide which is human peroxyredoxin.
. 配列番号 2のァミノ酸配列を有するヒ ト · ペルォキシレドキシン 2 相同体である請求項 1の血管炎抗原ぺプチド。 2. The vasculitis antigen peptide according to claim 1, which is a human peroxyredoxin 2 homolog having the amino acid sequence of SEQ ID NO: 2.
求項 1の抗原べプチドをコードするポリヌクレオチド  A polynucleotide encoding the antigenic peptide of claim 1
配列番号 1の塩基配列を有し、 請求項 2のヒ ト ·ペルォキシレドキ シン 2相同体をコードする請求項 3のポリヌクレオチド。  4. The polynucleotide according to claim 3, which has the nucleotide sequence of SEQ ID NO: 1, and encodes the human peroxyredoxin 2 homologue according to claim 2.
請求項 3のポリヌクレオチドを PCR増幅するためのプライマーセッ 卜 σ Primer set Bok σ for the claim 3 of the polynucleotide PCR amplification
請求項 1の抗原べプチドと結合する抗体。  An antibody that binds to the antigenic peptide of claim 1.
請求項 6の抗体とは異なるェピトープと結合する抗体。  An antibody that binds to a different epitope than the antibody of claim 6.
被験者から採取した生体試料中の前記発明（1)の抗原べプチドと結 合する抗体を検出し、 生体試料中にその抗体が健常者よりも多く存 在する被験者を血管炎患者または血管炎ハイリスク者と判定するこ とを特徴とするヒ ト血管炎診断方法。  An antibody that binds to the antigenic peptide of the invention (1) is detected in a biological sample collected from a subject, and the subject in which the antibody is present in a biological sample in a greater amount than a healthy subject is identified as a vasculitis patient or a vasculitis high. A method for diagnosing human vasculitis, characterized by determining that the person is at risk.
抗原べプチドを固定化した担体上において被験者から採取した生体 試料中の抗体と抗原べプチドとの結合を試験する請求項 8の診断方 法。  9. The diagnostic method according to claim 8, wherein the binding between the antibody in the biological sample collected from the subject and the antigen peptide is tested on a carrier having the antigen peptide immobilized thereon.
0 . 被験者から採取した生体試料中の請求項 6の抗体と結合する抗原 ぺプチドを検出し、 生体試料中にその抗原ぺプチドが健常者より も多く存在する被験者を血管炎患者または血管炎ハイリスク者と 判定することを特徴とするヒ ト血管炎診断方法。  0. An antigen peptide that binds to the antibody of claim 6 is detected in a biological sample collected from a subject, and a subject with a greater amount of the antigen peptide in the biological sample than a healthy subject is identified as a vasculitis patient or a vasculitis high. A method for diagnosing human vasculitis, characterized by determining that the subject is at risk.
. 請求項 6の抗体を固定化した担体上において、 抗体と抗原べプチ ドの結合を試験する請求項 1 0の診断方法。  10. The diagnostic method according to claim 10, wherein the binding between the antibody and the antigen peptide is tested on a carrier on which the antibody according to claim 6 is immobilized.
2 . 被験者から採取した生体試料における請求項 3のポリヌクレオチ ドの存在量を試験し、 ポリヌクレオチドの存在量が健常者のそれ らと比較して多い被験者を血管炎患者または血管炎ハイリ スク者 と判定することを特徴とするヒ ト血管炎診断方法。2. The polynucleotide according to claim 3 in a biological sample collected from a subject. A method for diagnosing human vasculitis, which comprises examining the abundance of a polynucleotide, and determining a subject having a higher abundance of a polynucleotide compared to that of a healthy subject as a vasculitis patient or a high-risk vasculitis patient.
3 少なく とも以下の要素 ： 3 At least the following elements:
(a) 請求項 1の抗原ペプチド； および  (a) the antigenic peptide of claim 1; and
(b) 請求項 1 の抗原べプチドと結合する抗体に特異的に結合す る標識化抗体  (b) a labeled antibody that specifically binds to the antibody that binds to the antigenic peptide of claim 1
からなることを特徴とする血管炎診断キッ ト。  A diagnostic kit for vasculitis, comprising:
4 少なく とも以下の要素 ：  4 At least the following elements:
(a) 請求項 1の抗原ペプチドを固定化した担体 ； および  (a) a carrier on which the antigenic peptide of claim 1 is immobilized; and
(b ) 請求項 1の抗原べプチドと結合する抗体に特異的に結合す る標識化抗体  (b) a labeled antibody that specifically binds to the antibody that binds to the antigenic peptide of claim 1
からなることを特徴とする血管炎診断キット。  A diagnostic kit for vasculitis, comprising:
5 少なく とも、 請求項 6の抗体おょぴノまたは標識化した請求項 7 の抗体を含むことを特徴とする血管炎診断キッ ト。  5 A kit for diagnosing vasculitis, comprising at least the antibody of claim 6 or a labeled antibody of claim 7.
6 少なく とも以下の要素 ：  6 At least the following elements:
(a) 請求項 6の抗体； および  (a) the antibody of claim 6; and
(b) 標識化した請求項 7の抗体  (b) the labeled antibody of claim 7
からなることを特徴とする血管炎診断キッ ト。  A diagnostic kit for vasculitis, comprising:
7 少なく とも以下の要素 ：  7 At least the following elements:
(a) 請求項 6の抗体を固定化した担体； および  (a) a carrier on which the antibody of claim 6 is immobilized; and
(b) 標識化した請求項 7の抗体  (b) the labeled antibody of claim 7
からなることを特徴とする血管炎診断キッ ト。  A diagnostic kit for vasculitis, comprising:
8 血液に不溶性の担体に固定化されたヒ トのペルォキシレドキシン またはその抗原決定基を含む断片を含む、 血管炎の治療剤。 8 A therapeutic agent for vasculitis, comprising human peroxyredoxin or a fragment containing an antigenic determinant thereof, which is immobilized on a carrier insoluble in blood.
9 次の工程を含む、 血管炎の治療または予防方法。 (a) 患者から採取した血液または抗体を含むその分画を、 血液に 不溶性の担体に固定化されたヒ トのペルォキシレドキシン またはその抗原決定基を含む断片に接触させる工程、 およ ぴ 9 A method for treating or preventing vasculitis, comprising the following steps. (a) contacting blood or a fraction thereof containing antibodies collected from a patient with human peroxyredoxin or a fragment thereof containing an antigenic determinant immobilized on a blood-insoluble carrier; andぴ
(b) 工程（a)の後に当該血液またはその分画を、 患者に戻す工程, 抗体を含む分画が血漿である請求項 19に記載の方法。  20. The method according to claim 19, wherein (b) returning the blood or a fraction thereof to the patient after step (a), and the fraction containing the antibody is plasma.
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