WO2004094638A1 - Antigene de la vasculite et technique de diagnostic de vasculite - Google Patents

Antigene de la vasculite et technique de diagnostic de vasculite 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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Prior art keywords
antibody
vasculitis
subject
peptide
antigen peptide
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PCT/JP2004/005729
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English (en)
Japanese (ja)
Inventor
Tomohiro Kato
Kusuki Nishioka
Shoichi Ozaki
Rie Karasawa
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St. Marianna University, School Of Medicine
Medical And Biological Laboratories Co., Ltd.
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Priority to JP2005505770A priority Critical patent/JPWO2004094638A1/ja
Publication of WO2004094638A1 publication Critical patent/WO2004094638A1/fr

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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.

Abstract

La présente invention concerne un peptide antigénique humain de la vasculite qui est une peroxyredoxine et une technique de diagnostic de vasculite qui consiste à examiner la présence ou l'absence d'un anticorps se liant au peptide antigénique susmentionné dans le sérum d'un sujet.
PCT/JP2004/005729 2003-04-21 2004-04-21 Antigene de la vasculite et technique de diagnostic de vasculite WO2004094638A1 (fr)

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WO2008004665A1 (fr) * 2006-07-06 2008-01-10 Fatigue Science Laboratory Inc. Procédé et appareil pour tester un cancer, un lupus érythémateux systémique (sle) ou un syndrome d'anticorps antiphospholipide à l'aide de rayons proches de l'infrarouge
FR2942541A1 (fr) * 2009-02-25 2010-08-27 Assist Publ Hopitaux De Paris Procede de diagnostic d'une vascularite

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FR2798672A1 (fr) * 1999-09-17 2001-03-23 Commissariat Energie Atomique Formes acides des peroxyredoxines et leur utilisation comme moyen de diagnostic

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008004665A1 (fr) * 2006-07-06 2008-01-10 Fatigue Science Laboratory Inc. Procédé et appareil pour tester un cancer, un lupus érythémateux systémique (sle) ou un syndrome d'anticorps antiphospholipide à l'aide de rayons proches de l'infrarouge
JP5047962B2 (ja) * 2006-07-06 2012-10-10 株式会社疲労科学研究所 近赤外光を用いたガン、全身性エリテマトーデス（ｓｌｅ）又は抗リン脂質抗体症候群に関する検査・診断装置の作動方法
FR2942541A1 (fr) * 2009-02-25 2010-08-27 Assist Publ Hopitaux De Paris Procede de diagnostic d'une vascularite
WO2010097553A2 (fr) 2009-02-25 2010-09-02 Assistance Publique - Hôpitaux De Paris Procédé de diagnostic d'une vascularite
WO2010097553A3 (fr) * 2009-02-25 2010-10-21 Assistance Publique - Hôpitaux De Paris Procédé de diagnostic d'une vascularite
EP2653869A2 (fr) 2009-02-25 2013-10-23 Assistance Publique - Hôpitaux de Paris Procédé de diagnostic d'une vascularite

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