[go: up one dir, main page]

WO2001090195A1 - Methode de dosage immunologique pour phospholypase a2 de type x - Google Patents

Methode de dosage immunologique pour phospholypase a2 de type x Download PDF

Info

Publication number
WO2001090195A1
WO2001090195A1 PCT/JP2000/008198 JP0008198W WO0190195A1 WO 2001090195 A1 WO2001090195 A1 WO 2001090195A1 JP 0008198 W JP0008198 W JP 0008198W WO 0190195 A1 WO0190195 A1 WO 0190195A1
Authority
WO
WIPO (PCT)
Prior art keywords
type
spla
cancer
antibody
polypeptide
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/JP2000/008198
Other languages
English (en)
Japanese (ja)
Inventor
Kohji Hanasaki
Keiichi Imagawa
Keiichi Masuta
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shionogi and Co Ltd
Original Assignee
Shionogi and 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 Shionogi and Co Ltd filed Critical Shionogi and Co Ltd
Priority to AU2001214187A priority Critical patent/AU2001214187A1/en
Priority to PCT/JP2001/004267 priority patent/WO2001090196A1/fr
Priority to AU58798/01A priority patent/AU5879801A/en
Priority to JP2001587007A priority patent/JP4726031B2/ja
Publication of WO2001090195A1 publication Critical patent/WO2001090195A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)

Definitions

  • the present invention relates to an antibody that specifically recognizes a part of X-type phospholipase A 2 (X-type sPLA 2 ); a method for measuring active X-type SPLA2 using the antibody; and a diagnostic kit containing the antibody.
  • X-type sPLA 2 X-type phospholipase A 2
  • Phospholipase 2 (PLAj; EC 3.1.1.4) is a generic name for phospholipidases that hydrolyze the 2-acyl ester bond of 3-s phosphoglyceride.
  • PLA 2 is involved in the digestion of dietary phospholipids and the formation and metabolism of biological membrane phospholipids, and also produces lipid media such as prostaglandin, leukotriene, platelet activating factor (PAF), and lysophospholipids.
  • Human type X sPLA 2 on the basis of the sPLA! Related sequences from DNA databases, which is black-learning Ri by fetal lung tissue (Cupillard, et, J. Biol. Chem., 272 , 15745-15752 (1997)).
  • Human type X sPLA the gene is located on the first 6 on chromosome unlike other sPLA 2.
  • Human type X sPLA ⁇ proteins, the most acidic in the sPLA 2 family (pi 5.3), - contains a linked sugar chain binding site capable.
  • This sPLA 2 has a 1 6 cysteine residues in the molecule, contain characteristic intramolecular disulfide bridges present in each type IB sPLA 2 and ⁇ ID / IIE type sPLA 3 ⁇ 4 Te to base. Furthermore, having a unique force Rupokishiru group terminal extension structure IIA ID / IIE type sPLA 2.
  • expression of type X sPLA ⁇ MA 1.5 kb
  • physiological type X sPLA 2 is unknown detail with pathological features, the localization of the expression site, the enzyme is estimated be involved in the regulation of the immune system and inflammatory conditions Is done.
  • An object of the present invention X-type part of sPLA 2 antibodies specifically recognizing; to provide a diagnostic kit comprising said antibody; Measurement method of active type X sPLA 2 with antibody .
  • the present inventors have, have extensive Ken ⁇ with the aim to elucidate the physiological functions of the human X-type sPLA 2 Was, but in the process, the X-type sPLA 2, the existence and proform inactive form having a propeptide sequence N- terminus, two molecular species of active that occurs the propenyl peptide portion is cut The amino acid sequence of the propeptide portion cleaved during the activation process was identified.
  • the present inventors have found a measuring method for specifically detecting the concentration of the activated X-type sPLAr by measuring the amount of the propeptide or the amount of the active X-type sPLA ⁇ itself, and completed the present invention. That is, the present invention
  • the antibody according to (1) which specifically recognizes a polypeptide consisting of the amino acid from Gly at position 1 to Asp at position 123 in the amino acid sequence of SEQ ID NO: 3; 4) The antibody according to any one of (1) to (3) above, which is a polyclonal antibody;
  • the therapeutic agent PLA 2 related diseases which comprises the antibody according to (3);
  • polypeptide comprising an amino acid from Gly at position 1 to Asp at position 123 in the amino acid sequence of SEQ ID NO: 3;
  • a kit for cancer diagnosis comprising a reagent capable of detecting the polypeptide according to (6) or (7) above;
  • a kit for diagnosing Alzheimer's disease comprising a reagent capable of detecting the polypeptide of (6) or (7) above;
  • a kit for diagnosing cirrhosis comprising a reagent capable of detecting the polypeptide according to (6) or (7) above;
  • X-type sPLA There are three types of X-type sPLA; depending on the degree of maturation, prebub type X-type sPLA pro-type X-type sPLA 2 and active type X-type sPLA 2 (also called mature type X-type sPLA 2 ).
  • type X sPLA Immediately after translation from mRNA, type X sPLA; Produced as a mold.
  • the Purebu port type X type sPLA 2 in the amino acid sequence set forth in SEQ ID NO: 3 means a Poripe petit de consisting Amino acids to Asp 1 2 3 position from Met one 3 2 position.
  • the signal peptide is a polypeptide consisting of an amino acid from Met at position 132 to Gly at position 112 in the amino acid sequence of SEQ ID NO: 3.
  • Pro X-type S PLA 2 occurs connection by that signal peptide from Purebu port type is disconnected.
  • the pro-form X-type sPLAi refers to a polypeptide consisting of the amino acids from Glu at position 11 to Asp at position 123 in the amino acid sequence of SEQ ID NO: 3. Furthermore, pro-type X sPLA!
  • a fragment composed of a signal peptide and a propeptide is sometimes referred to as a prepropeptide.
  • the prepropeptide means a polypeptide consisting of the amino acid from Met at position 132 to Arg at position 11 in the amino acid sequence shown in SEQ ID NO: 3 (FIG. 1).
  • it means a propeptide or active X-type sPLAi.
  • a polypeptide j in "(6) or (7), means Puropepu tides or active type X sPLA 2.
  • active X-type sPLA 2 immunological means Intended to be measured by Things.
  • pro-type X sPLA ⁇ produces a pair of activated X-type sPLA;
  • the present inventors have found that by specifically measuring propenyl peptide that is produced equimolar mass and active type X sPLA 2 in the course of active type X sPLAi or activation, measurement of the active X-type sPLA 2 Completed the method.
  • an antibody specifically recognizing a polypeptide consisting of amino acids from Glu at position 11 to Arg at position 1 in the amino acid sequence of SEQ ID NO: 3 Means an antibody that specifically recognizes it.
  • Specific recognition of propeptide means that it reacts with propeptide but does not react with active X-type sPLA! Or pro-X type sPLA; which has a peptide fragment. I do.
  • amino acid sequence depicted in SEQ ID NO: 3 wherein position 1 polypeptide antibodies specifically recognizing consisting amino acids from Gl y to 1 2 3 position of the Asp and the active type X sPLA 2 It means an antibody that specifically recognizes it.
  • 'And' active type X sPLA 2 specifically recognizing ", although reacts with active X-type SPLAs, means that does not react with propenyl peptide Ya proform X type sPLA 2. “No reaction” means “the antigen and the antibody do not bind” in the ELISA method. Moreover, the propeptide or active type X sPLA 2 was used to prepare these antibodies can be used as the "standard antigen” to generate a standard curve. By using the antibodies of the present invention, it is measured only active type X sPLA 2 becomes possible.
  • the secret type is involved in the release of fatty acids (eg, arachidonic acid) from phospholipids present in cell membranes and lipoproteins. Excessive release of fatty acids and the production of various lipid media throughout their metabolism contribute to diseases such as septic shock, adult respiratory distress syndrome, inflammation, trauma, bronchial asthma, allergic rhinitis, and rheumatoid arthritis. Become.
  • PHA 2 related disease in the present invention means those diseases.
  • Antibodies to activated type X sPLA 2 of the present invention are useful for the treatment of these diseases.
  • the use of the measurement method of the present invention enables the diagnosis of these diseases caused by activated X-type sPLA ⁇ .
  • the present invention provides a "method of measuring active type X sPLA 2". Furthermore, the present inventors have found that, by immunohistochemical analysis, colon cancer tissue of colon cancer patients, lung cancer tissue of lung cancer patients, liver tissue of liver cancer patients, stomach tissue of gastric cancer patients, stomach tissue of kidney cancer patients, X-type sPLAz is more prominent in gallbladder tissue of gallbladder cancer patients, prostate tissue of prostate cancer patients, brain tissue of kidney cancer patients, cerebral tissue of Alzheimer's disease patients, and liver tissue of cirrhosis patients as compared to normal human tissues was confirmed to be highly expressed.
  • the antibodies of the phospholipid or the present invention is a substrate for sPLA 2 is used as "reagents", by confirming the activity or expression of type X sPLA 2, cancer, it is possible to diagnose Alzheimer's disease or cirrhosis.
  • the present invention provides a "kit for diagnosing cancer", a "kit for diagnosing Alzheimer's disease", or a "kit for diagnosing cirrhosis”.
  • Each diagnostic kit contains at least a reagent capable of detecting the polypeptide of the present invention.
  • the reagent include a phospholipid which is a substrate of SPLA2 and the antibody of the present invention.
  • Examples of the detection method include a sedimentation reaction method, an ELISA method, an RIA method, a western blotting method, and the like.
  • an antibody is reacted with an appropriately diluted patient serum, washed, washed with a secondary antibody, a peroxidase-labeled anti-Egret IgG antibody, etc., and allowed to react.Then, a peroxidase substrate is added. By developing the color and measuring the absorbance at 450 nm, the polypeptide of the present invention can be detected.
  • the kit of the present invention includes, in addition to the antibody of the present invention, various reagents such as a coloring reagent, a reaction stopping reagent, a standard antigen reagent, and a sample pretreatment reagent as necessary.
  • various reagents such as a coloring reagent, a reaction stopping reagent, a standard antigen reagent, and a sample pretreatment reagent as necessary.
  • An appropriate reagent according to the measurement method can be appropriately selected and can be attached to the kit of the present invention.
  • cancer especially colon cancer, lung cancer, and liver It will be possible to conduct mass screening for early detection of organ cancer, stomach cancer, kidney cancer, gallbladder cancer, prostate cancer or Teng's cancer, Alzheimer's disease or cirrhosis.
  • the present invention also provides a "screening method" for diagnosis. BRIEF DESCRIPTION OF THE FIGURES
  • Figure 1 is a diagram showing the relationship between Purebu port type X-type sPLA flop D-type X-type sPLA ⁇ and active type X sPLA 2.
  • FIG. 2 is a diagram showing the enzymatic activities of mouse activated X-type sPLA 2 , mouse pro-type X-type sPLA 2, and tryptic quenched pro-type X-type sPLA ⁇ .
  • M, P and P + T it it active, to agree taste has been pro-type X-type sPLA 2 digested with proform Oyobito trypsin.
  • FIG. 3 shows a standard curve in the ELISA method using the anti-X type sPLA ⁇ peptide antibody of the present invention.
  • the present invention relates mainly measurement of active type X sPLA 2 with propeptide.
  • DNA encoding the type X sPLA 2 is based on the sequence information taught by the present invention generally It can be easily produced and obtained by genetic engineering techniques (see Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989), etc.). Specifically from an appropriate origin which X type sPLA 2 is expressed, the cDNA library one was prepared according to a conventional method, using a suitable probe or antibody specific from the library one of the nucleotide sequence of X-type sPLA 2 Acad. Sci., USA., 78, 6613 (1981); Science, 22, 778 (1983) and the like can be used to select the desired clone.
  • CMA CMA-associated antigen-specific senor
  • various cells expressing type X sPLA 2 CMA-associated antigen-specific senor
  • separation of total MA, isolation and purification of mRNA, acquisition of cMA, and cloning thereof can all be carried out according to conventional methods.
  • cDNA libraries are commercially available, and in the present invention, such cDNA libraries, for example, various types of cMA libraries commercially available from Clontech can also be used.
  • the method for screening the DNA of X-type sPLA ⁇ from the cMA library is not particularly limited, and a usual method can be used. Specifically, for example, a method for selecting a cDNA clone corresponding to a protein produced by cDNA by immunoscreening using a protein-specific antibody, a probe that selectively binds to a target MA sequence Plaque hybridization, colony hybridization, and the like using these methods.
  • MA and the like chemically synthesized based on information on the base sequence of X-type sPLA ⁇ can be generally exemplified, but of course, the DNA itself and fragments of the present invention which have already been obtained are also good.
  • DNA encoding a type X sPLA 2 is conventional chemical methods, for example tricalcium E ester method (Narang et al., Met. Enzymol., 68, 90-108 (1979)) or phosphorus dihydrogen It can be synthesized by the ester method (Brown et al., Meth. Enzymol., 68, 109-151 (1979)).
  • X-type sPLA ⁇ protein according to the base sequence information of the X-type sPLA 2, genetic engineering proposed method (Science, 224, 1431 (1984 );... Biochem Biophys Res Comm, 130, 692 (1985);. Proc. Natl. Acad. Sci., USA., 80, 5990 (1983), etc.). More specifically, a gene encoding a desired protein is inserted into an appropriate vector. This vector is introduced into a host cell to prepare a transformant. By culturing the transformant, a recombinant protein can be obtained.
  • the eukaryotic cells include cells such as vertebrates and yeasts.
  • Examples of the vertebrate cells include COS cells (Cell, 23, 175 (1981)) which are monkey cells, and chicks. Hamster ovary cells are often used.
  • a vector having a promoter, a MA splice site, a polyadenylation site, a transcription termination sequence, and the like, which is usually located upstream of the gene to be expressed can be used. May be. It is an example of the expression vector, for example, P SV2dhfr carrying early promoter of SV40 (Mol. Cell. Biol. , 1, 854 (1981)) or the like can be exemplified. As eukaryotic microorganisms, yeasts are generally used, and Saccharomyces yeasts can be used. As an expression vector for the yeast, for example, pAM82 (Proc. Natl. Acad.
  • Escherichia coli and Bacillus subtilis are commonly used as prokaryotic hosts. These When a host is used, for example, a plasmid vector capable of replicating in the host bacterium is used, and a promoter, an SD sequence, and a protein are arranged upstream of the gene so that a desired gene can be expressed in the vector. It is preferable to use an expression plasmid having an initiation codon required for the initiation of synthesis. As the above host, E. coli K12 strain or the like is used.
  • PBR322 and its improved vector are often used as vectors, but not limited thereto, and various known strains and vectors can also be used.
  • the promoter for example, trp promoter, lpp promoter, lac promoter, PL / PR open motor and the like can be used.
  • a method for introducing a desired recombinant MA into a host cell and a transformation method using the same various general methods can be adopted.
  • the obtained transformant can be cultured according to a conventional method, and the desired protein is produced by the culture.
  • the medium used for the culture various types commonly used depending on the host cell can be appropriately selected and used, and the culture can be performed under conditions suitable for the host cell.
  • a transformant is prepared by introducing a vector containing the human X-type sPLAi gene of the present invention downstream of the pSVL SV40 late promoter into monkey-derived cells COS-7, and the transformant is present in 5% C02. lower, by culturing for 3 days at 37 ° C, recombinant human type X sPLA 2 evening protein can be produced.
  • Proteins can be separated by various separation procedures utilizing their physical and chemical properties (Biochemistry, 25 (25), 8274 (1986); Eur. J. Biochem., 163, 313 (1987), etc.). Can be purified. Examples of the method include salting out, centrifugation, osmotic shock, sonication, ultrafiltration, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, and high performance liquid chromatography. Examples thereof include liquid chromatography, dialysis, and combinations thereof.
  • Purified X-type sPLA 2 is for containing a proform X type SPLAi, (if example embodiment, subtilisin Endopurotea Ichize, etc. trypsin) Suitable proteases in the child cutting gives active type X sPLA ⁇ I can do it.
  • X-type sPLA ⁇ protein, or X-type sPLA 2 protein fragment e.g., propenyl Petit de
  • X-type sPLA ⁇ protein, or X-type sPLA 2 protein fragment e.g., propenyl Petit de
  • a peptide synthesizer Preparation of Antibodies Against Protein of the Present Invention
  • An antibody against the protein of the present invention is produced by the following method.
  • type X sPLA 2 Based on the amino acid sequence of type X sPLA 2, produced conventional synthetic peptides and synthesized in peptide synthesizer, bacteria transformed with a vector expressing the type X sPLA 2, yeast, insect cells, and the like animal cells
  • the purified proteins are purified by ordinary protein chemistry methods and these are used as immunogens.
  • immunize animals by an appropriate method according to the method described in Antibodies; A Laboratory Manual, Lane, H. D. et al., Edited by Cold Spring Harber Laboratory Press, New York, 1989.
  • a polyclonal antibody that specifically recognizes a protein serving as an antigen can be easily prepared and purified. Animals such as mice, rats, hamsters, and egrets can be immunized to produce polyclonal antibodies derived from their sera.
  • Lymphocytes are removed from the spleen or lymph node of a mouse rat immunized with the above-mentioned immunogen, fused with myeloma cells, and subjected to Kohler and Mil stein's method (Nature, 256, 495-497 (1975)). )
  • a monoclonal antibody can be produced from the hybridoma. Pair propenyl peptide or active type X sPLA 2, for example, by the following steps You can obtain a monoclonal antibody that:
  • the measurement method of the present invention detects the amount of an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of antigen (propeptide or active type X sPLA 2 ) in a test solution by chemical or physical means. Any measurement method may be used as long as the measurement method is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, a competition method, an immunometric method, and a sandwich method can be used.
  • a chemical bond usually used for immobilizing a protein or an enzyme can be used.
  • the carrier insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used. Radiolabels, enzymes, and fluorescent substances are used as labeling agents. Radioisotopes, 1 2 5 I, 3 H , etc. H G is used.
  • the enzyme peroxidase, 5-galactosidase, / 3-darcosidase, alkaline phosphatase, etc. are used.
  • CMA encoding the human type X sPLA 2 is, as ⁇ the human placenta cMA, was obtained Ri by the PCR method. The following primers were used for PCR.
  • hGX-S 5'-ctgtgtacgcgtccatgctgctcctgctactgccgtc-3 '(SEQ ID NO: 5)
  • GX-AS 5'-tcaagtgcggccgctcagtcacacttgggcgcgtcc-3 '(system No. 6)
  • hGGX_S has a Kozak sequence and a Mlul recognition site.
  • HGX-AS also has a Notl recognition site.
  • PCR was performed for 35 cycles at 30 degrees at 94 degrees, 5 seconds at 50 degrees, and 2 minutes at 72 degrees.
  • the PCR amplified fragment was cut with Mlul and Notl, and introduced into pBS-SK (-).
  • the X-type sPLA “HisTa-form” with 6 His residues added to the carboxyl group terminus can be obtained from the hGX-S and hGX-H6AS primers (5,- -3 ':.
  • SEQ ID NO: 7 was used, was constructed by PCR PCR amplified fragment was cut with Notl and Xhol, the corresponding substituted with site PCR sequence of regions ⁇ in the X-type sPLA 2 plasmid. . after checking, the cDNA was ⁇ the SR- alpha promoter downstream of mammalian cell expression vector in example 2 human type X sPLA 8 - expression of HisTag recombinant proteins, purification and characterization
  • This 293T clone was cultured in a medium containing 10% fetal calf serum, and a culture supernatant containing X-type sPLA ⁇ -HisTag-expressing protein was prepared.
  • the main culture supernatant was equilibrated with 20 mmol / L sodium phosphate buffer (pH 7.4) containing 10 nimol / L imidazole and 0.5 mol / L sodium chloride, and Ni 2 + -charged chelating Sepharose fast fl ow column (Amersham Pharmacia Biotech).
  • the sPLA 2 -HisTag protein was bound to this column; the sPLA 2 -HisTag protein was converted to 20 mmol / L sodium phosphate buffer (pH 7.4) containing 500 mmol / L imidazole and 0.5 mol / L sodium chloride. Eluted.
  • the fraction containing the obtained X-type sPLA 2 -HisTag protein was dialyzed against a 20-mol / L Tris-HCl buffer containing 1 mmol / L phenylmethyl sulfonic acid, and a HiTrap Q column. (Amersham Pharmacia Biotech).
  • the bound protein was eluted with a gradient of sodium chloride from 0 to 0.5 mol / L, and the fraction containing the sPLA 2 -HisTag type X protein was then eluted, followed by a reversed-phase HPLG column (Cosmosil, 5C18 300AR, 4.6 x 150 mm, Nacalai Tesque). Elution was performed with a concentration gradient of acetonitrile from 20 to 95% in 0.05% trifluoroacetic acid, and the resulting fraction was measured for chromogenic PLA 2 activity.As a result, X-type sPLA 2 -His Tag was 43% Was eluted with acetonitrile.
  • the antiserum was prepared and purified the IgG fraction using a HiTrap Protein G- column (Amersham Pharmacia Biotech). The specificity and titer of the antiserum and the purified antibody were measured by the following ELISA method.
  • anti-X type sPLA 2 antibody thus obtained contained human X-type sPLAi was added HisTag although to recognize, did not react with type IB or I IA type sPLA 2.
  • the prepared antibody did not react with His-tagged protein which is not related to X-type sPLA ⁇ . Therefore, Hi sTag additional part, it became clear that not Epito one flop anti X type sPLA 2 antibody.
  • Enzymatic activity obtained by adding HisTag is dose-dependently inhibited, in a concentration of 100 ⁇ g / nil, the other five sPLAi activity the effect was not observed, only the enzymatic activity of type X sPLA 2 was impaired about 90% inhibitory. From the above results, anti-type X sPLA ⁇ antibody preparation proved to be specific for Tan Pak portion of human type X sPLA 2.
  • Example 4 Expression, purification and characterization of recombinant type X sPLA g protein
  • the CH0 cells that the enzyme was stable expression was prepared as described above.
  • the CH0 cells were transformed with Dulbecco's modified serum containing 10% fetal calf serum.
  • the cells were changed to serum-free PM-1000 medium (Eiken, Japan), and the cells were further cultured for 3 days.
  • the resulting culture supernatant was added to Afi two tee column coupled with the anti-type X sPLA 2 antibody.
  • This column is an anti-type X sPLA 2 I gG (30 mg) , HiTrap N- human Dorokishisakushi N'i Mi de-activated column according to a conventional method; prepared by coupling the (5 mL Amersham Pharmaci a B i otech). After washing the column with sodium phosphate buffer (pH 7.4), the bound proteins were eluted with 0.1 mol / L glycine-HCl (pH 1.7). This eluted fraction was applied to the above-mentioned reversed-phase HPLC column, and separated with a gradient of 20 to 40% acetonitrile in 0.1% trifluoroacetic acid.
  • N-glycosidase F treatment it is aggregated into two molecular species (14-kDa and 13-kDa), and as a result of their N-terminal amino acid sequence analysis, a majority of 13-kDa protein (GI LELAGT: SEQ ID NO: 8) is (in the amino acid sequence set forth in SEQ ID NO: 3, polypeptides consisting of Asp 1 2 3 position from position 1 Gly) predicted mature of human type X sPLA 3 ⁇ 4 a while, proteins few 14-kDa a peptide consisting of 11 residues at the N-terminus of the mature type X sPLA 2: in the amino acid sequence set forth in (EASRILRVHRR SEQ ID NO: 9) is added professional type (SEQ ID NO: 3, - 1 from 1-position of Glu 1 2 3 (A polypeptide comprising Asp). Therefore, 15 ⁇ 18- kDa Niwata connexion molecular species showing a broad molecular weight was considered as X-type
  • Each sPLA 2 protein for enzymatic activity (IB type, I IA type sPLA 2 and HPLC 3 fractions of human type X sPLA 2), each consisting of Li phospholipids and 3 mmol / L Dokishikoru acid of 1 mmo l / L It was measured under mixed micelle conditions.
  • the mature form of X-type sPLA ⁇ (fractions 2 and 3) showed the highest activity against phosphatidylcholine bearing arachidonic acid at the sn-2 position .
  • IB-type and I IA type sPLA 2 showed the highest activity against phosphatidylglycerol for holding Orein acid at the 2-position.
  • the present inventors have found that in the process of purification of recombinant human type X sPLA 2 protein, mature type X sPLA 2 protein N-terminal 11 amino consisting of the amino acid propenyl peptide is added pro X-type sPLA
  • the presence of 2 and the substrate specificity analysis indicated that the pro-type enzyme may have much lower enzymatic activity than the mature form.
  • the signal sequence Using SignalP computer analysis to predict the cleavage site (Nielsen et al., Protein Eng., 12, 3-9 (1999)), the site that is most susceptible to signal peptide cleavage is the N-terminus of the mature protein.
  • the 11 amino acid residue was proved to be a propeptide by showing that it was between the eleventh and eleventh position earlier. Therefore, it was shown that cleavage of the propeptide from the pro-form by a subtilisin-like or trypsin-like protease is essential for the expression of the maximal enzymatic activity of type X sPLA ⁇ . by measuring the production amount, it becomes possible to predict the production of activity type X type sPLA 2 occurring in pairs.
  • Specimens of healthy adult colon tissue and colon cancer tissue from colon cancer patients were purchased from Biochain In (Sa Leandro, CA). Slide of tissue section, remove the paraffin wax, after removing the main evening endogenous Peruokishi evening over peptidase reacted for 30 minutes in Nord containing 0.3% H 2 0 2, 20 minutes with 5% normal turbocharger formic serum Treated. Then, in PBS containing 0.1% bovine serum albumin, anti-X type sPLA 2 antibody (6 ⁇ g / mL) and 14 hours to reaction at 4 ° C.
  • the cells were reacted with a goat anti-Peacock IgG antibody conjugated with biotin for 30 minutes, and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, the 200 JUL g / mL of di ⁇ amino benzidine and 50 nmol / L Tris- HC1 (pH 7.6) by Ri Peruokishidaze activity to react for 10 minutes in a buffer containing 0.006% H 2 0 3 ⁇ 4 coloration type X sPLA 2 in and tissue specimens were visualized. Cell nuclei were counterstained by reaction with 0.4% hematoxylin aqueous solution.
  • X-type sPLA 2 positive signal was detected as deposits dark brown di ⁇ amino benzylidene den.
  • Neutralization experiments X-type sPLA 3 ⁇ 4 signal, prior to the addition of antibody to slide, was purified X-type sPLA 2 protein and (60 pi g / mL) carried out by reacting for 2 hours.
  • a positive signal is X-type sPLA 2
  • X-type sPLA 2 is hardly detected in normal colon tissues was detected strongly in cancer cells of colon adenocarcinoma tissues.
  • These signals are type X sPLA 2 It was confirmed to be specific to type X sPLA 2 since disappeared by the addition of protein. In addition, these positive signals were not observed in IgG prepared from non-immunized egrets. These results, in colorectal cancer suggested that the expression of type X sPLA 2 protein is significantly high expression.
  • Specimens of healthy adult lung tissue and lung tissue from lung cancer patients were purchased from Biochain Inc. (San Leandro, CA). Slide of tissue section, except take paraffin Wa Tsu box, after removing the endogenous Peruokishitaze reacted methanol for 30 minutes containing 0.3% H 2 0 2, was treated with 5% normal turbocharger formic serum for 20 minutes . Then, in PBS containing 0.1% bovine serum albumin, anti-X type sPLA 2 antibody (6 JUL g / mL) and 14 hours, the mixture was reacted at 4 ° C.
  • the cells were reacted with a goat anti-Peacock IgG antibody conjugated with biotin for 30 minutes, and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 ⁇ g / mL 50 mmol / L Tris-HCl (H 7.6) containing Jiamino base Njijin and 0.006% H 2 0 2 in by reaction for 10 minutes in buffer and colored the Peruokishi Daze active tissue the X-type S PL in the sample was visualized. Cell nuclei were counterstained by reaction with an o.4% hematoxylin aqueous solution.
  • Type X SPLA2-positive signals were detected as dark brown diaminobenzidene deposits.
  • the neutralization experiment of the X-type sPLA 2 signal was performed by reacting the purified X-type sPLA, protein (0 jg / mL) for 2 hours before adding the antibody to the slide.
  • Specimens of healthy adult liver tissue and lung tissue from liver cancer patients were purchased from Biochain Inc. (San Leandro, CA). The slide of the tissue section was treated with methanol containing 0.3% 02 for 30 minutes to remove endogenous peroxidase after removing paraffin wax and then treated with 5% normal goat serum for 20 minutes. Then, in PBS containing 0.5 calf serum albumin, and 14 hours pile type X sPLA 3 ⁇ 4 antibody (6 JUL g / mL), and allowed to react at 4 ° C.
  • the cells were reacted with a goat anti-Peacock IgG antibody conjugated with biotin for 30 minutes, and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 ju g / mL 50 mmol / L Tris-HCl (pH 7.6) containing Jiamino base Njijin and 0.006% H 2 0 2 in by reaction for 10 minutes in buffer and colored the Peruokishi Daze active tissue X-type sPLA 2 in the specimen was visualized. Cell nuclei were counterstained by reaction with 0.4% hematoxylin aqueous solution.
  • X-type sPLA 2 positive signal was detected as deposits dark brown di ⁇ amino benzylidene den.
  • Neutralization experiments X-type sPLA 2 signal, prior to the addition of antibody to slide was performed purified X-type sPLA! Protein and (60 ig / mL) by reacting for 2 hours.
  • Specimens of healthy adult stomach tissue and lung tissue from gastric cancer patients were purchased from Biochain Inc. Leandro, CA). Slide of tissue section, except take paraffin WAX, after removing the endogenous Peruokishitaze reacted methanol Ichiru to 3 0 minutes containing 0.3% H 2 0 2, was treated with 5% normal turbocharger formic serum for 20 minutes . Then, in PBS containing 0.1% bovine serum Alp Min, anti X type sPLA 2 antibody (6 JUL g / mL) and 14 hours, the mixture was reacted at 4 ° C.
  • the cells were reacted with a goat anti-Peacock IgG antibody conjugated with biotin for 30 minutes, and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 JUL g / m coloration organized Peruokishi Daze activity by reacting for 10 minutes at 50 mmol / L Tris-HCl ( pH 7.6) buffer containing Jiamino base Njijin and 0.006% H 2 0 2 of X-type sPLA! In the specimen was visualized. Cell nuclei were counterstained by reaction with 0.4% hematoxylin aqueous solution.
  • X-type sPLA 2 positive signal was detected as deposits dark brown di ⁇ amino benzylidene den.
  • the experiment for neutralizing the X-type sPLA 2 signal was performed by reacting the purified X-type sPLA 2 protein (60 g / mL) for 2 hours before adding the antibody to the slide.
  • Specimens of healthy adult kidney tissue and lung tissue from kidney cancer patients were purchased from Biochain lnc. (San Leandro, CA). Slide of tissue section, except take paraffin Nwadzukusu, after removing the endogenous Peruokishi evening over peptidase reacted methanol for 30 minutes with 0.3% 0 2, were treated with normal catcher formic serum 5 20 minutes. Then, in PBS containing 0.1% bovine serum albumin, anti-X type sPLA 2 antibody (6 g / mL) and 14 hours, allowed to react at 4 ° C Was.
  • the cells were reacted with a goat anti-Peacock IgG antibody conjugated with biotin for 30 minutes, and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 jg / mL diaminobenzidine was reacted for 10 minutes in 50 mmol / L Tris-HCl (pH 7.6) buffer containing 0.006% to develop peroxidase activity, and the X-type in the tissue specimen was developed. sPLA 2 was visualized. Cell nuclei were counterstained by reaction with 0.4% hematoxylin aqueous solution.
  • Type X sPLA ⁇ -positive signals were detected as dark brown diaminobenzidene deposits.
  • the neutralization experiment of the X-type sPLA 2 signal was performed by reacting the purified X-type sPLA! Protein (60 g / mL) for 2 hours before adding the antibody to the slide.
  • X-type sPLAs positive signals were detected at low levels in glomerular mesangial cells in normal kidney tissues, but were strongly detected in cancer cells in kidney tissues from kidney cancer patients. These signals, it was confirmed to be specific to type X sPLA 2 since disappeared by the addition of X-type sPLA 2 protein. Moreover, these positive signals were not observed in IgG prepared from non-immunized egrets. These results suggest that renal cancer has remarkably high expression of type X sPLA ⁇ protein. Immunohistochemical analysis of human Tan ⁇ tissue with Example 1 1 Anti-X type sPLA 2 antibody
  • Specimens of healthy adult gallbladder tissue and lung tissue from gallbladder cancer patients were purchased from Biochain Inc. (San Leandro, CA). The slide from the tissue section was treated with 5% normal goat serum for 20 minutes after removing paraffin wax and reacting with methanol containing 0.3% H 2 O 2 for 30 minutes to remove endogenous peroxidase. Then, in PBS containing 0.1% bovine serum Alp Min, anti X type sPLA 2 antibody (6 ju g / mL) and 14 hours, the mixture was reacted at 4 ° C.
  • the cells were reacted with a goat anti-Peacock IgG antibody conjugated with biotin for 30 minutes, and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 50 containing 200 g / mL Jiamino base Njijin and 0.006% E l 0 1 of After reacting for 10 minutes in a mmol / L Tris-HCl (pH 7.6) buffer, the peroxidase activity was colored and the X-type sPLA ⁇ in the tissue specimen was visualized. Cell nuclei were counterstained by reaction with 0.4% hematoxylin ice solution.
  • X-type sPLA 2 positive signal was detected as deposits dark brown di ⁇ amino benzylidene den.
  • Neutralization experiments X-type sPLA 3 ⁇ 4 signal, prior to the addition of antibody to slide was performed purified X-type sPLA 3 ⁇ 4 protein and (60 ug / mL) by reacting for 2 hours.
  • Specimens of healthy adult prostate tissue and prostate tissue from prostate cancer patients were purchased from Biochain Inc. (SanLeandro, CA). The slide of the tissue section was treated with methanol containing OJXHsOi for 30 minutes to remove endogenous peroxidase, and then treated with 5% normal goat serum for 20 minutes. Then, it was reacted with anti-X sPLAs antibody (6 jug / mL) in PBS containing 0.1% bovine serum albumin for 4 hours at 4 ° C.
  • the cells were reacted with a goat anti-Peacock IgG antibody conjugated with biotin for 30 minutes, and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 ⁇ g / mL diaminobenzidine was reacted for 10 minutes in a 50 mmol / L Tris-HC1 (pH 7.6) buffer containing 0.006% to exhibit peroxidase activity, resulting in a color change in the tissue specimen. X-type sPLAi was visualized. Cell nuclei were counterstained by reaction with 0.4% hematoxylin aqueous solution.
  • Type X sPLA a positive signal was detected as dark brown diaminobenzidene deposits.
  • X-type sPLA ⁇ positive signal was hardly detected in normal prostate tissue, but was strongly detected in cancer cells in prostate tissue derived from prostate cancer patients. These signals, it was confirmed to be specific to type X sPLA 2 since disappeared by the addition of X-type sPLA 3 ⁇ 4 protein. In addition, these positive signals were not observed in IgG prepared from non-immunized egrets. These results, in prostate cancer suggested that the expression of type X sPLA 2 ⁇ white matter is significantly high expression.
  • Example 13 Immunohistochemical analysis of human spleen tissue using anti-X type sPLA and antibody
  • Specimens of healthy adult Teng tissue and arm tissue from Teng cancer patients were purchased from Biochain Inc. (SanLeandro, CA). Slide of tissue section, remove the paraffin wax, after removing the endogenous Peruokishi evening over peptidase reacted methanol for 30 minutes containing 0.3% 3 ⁇ 40 2, was treated with 5% normal turbocharger formic serum for 20 minutes. Then, in PBS containing 0.1% bovine serum albumin, anti-X type sPLA 3 ⁇ 4 antibody (6 ju g / mL) and 14 hours to reaction at 4 ° C.
  • the cells were reacted with a goat anti-Peacock IgG antibody conjugated with biotin for 30 minutes, and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, the reaction was performed for 10 minutes in a buffer containing 200 ⁇ g / mL diaminobenzidine and 50 mmol / L Tris-HCl (pH 7.6) containing 0.006% HA to develop peroxidase activity. X-type sPLA 2 in the specimen was visualized. Cell nuclei were counterstained by reaction with 0.4% hematoxylin aqueous solution.
  • X-type sPLA 2 positive signal was detected as deposits dark brown di ⁇ amino benzylidene den.
  • Neutralization experiments X-type sPLAj signal, prior to the addition of antibody to slide was performed purified X-type sPLA 2 protein and (60 ⁇ . G / mL) by reacting for 2 hours.
  • Specimens of healthy adult cerebral tissue and cerebral tissue from Alhamama patients were purchased from Biochain Inc. (SanLeandro, CA). Slide of tissue section, remove the paraffin Nwadzu box, after 0 ⁇ 3% Eta remove methanol is reacted for 30 minutes endogenous Peruoki sheet evening over peptidase containing 2 0 2, 5% of normal turbocharger formic serum Treated for 20 minutes. Then, in PBS containing 0.1% bovine serum albumin, anti-X type sPLA 2 antibody (6 ng / mL) and 14 hours, the mixture was reacted at 4.
  • the cells were reacted with a goat anti-Peacock IgG antibody conjugated with biotin for 30 minutes, and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 ⁇ . G / ml Jiamino base Njijin and 0.006% or 0 2 50 thigh ol / L Tris- HC1 (pH 7.6 ) containing buffer coloration said the I Ri Peruokishidaze activity to react for 10 minutes in X-type sPLA; in tissue specimens was visualized. Cell nuclei were counterstained by reaction with 0.4% hematoxylin aqueous solution.
  • X-type sPLA 2 positive signal was detected as deposits dark brown di ⁇ amino benzylidene den.
  • Neutralization experiments X-type sPLA 2 signal, prior to the addition of antibody to slide was performed purified X-type sPLA 2 protein and (60 ng / mL) by reacting for 2 hours.
  • X-type sPLA ⁇ -positive signals were hardly detected in normal cerebral tissues, and were partially detected in cerebral tissues derived from Alzheimer's patients, especially in senile plaques and neurofibrillary tangles ( Strongly detected in neurofibrillary tangle regions). These signals, it was confirmed to be specific to type X sPLA 2 since disappeared by the addition of X-type sPLA 2 protein. In addition, these positive signals were not observed in IgG prepared from non-immunized egrets. From the above results, Al ⁇ haima In one patient brain it suggested that the expression of type X sPLA 2 protein is significantly high expression.
  • Example 15 Immunohistochemical analysis of human liver cirrhosis using anti-X type sPLA ; antibody
  • Specimens of healthy adult liver tissue and liver tissue from cirrhosis patients were purchased from Biochain Inc. (SanLeandro, CA). The slide of the tissue section was treated with 5% normal goat serum for 20 minutes after removing paraffin wax and reacting with methanol containing 0.3% 02 for 30 minutes to remove endogenous peroxidase. Then, in PBS containing 0.1% bovine serum albumin, anti-X type sPLA 2 antibody (6 J g / mL) and 14 hours to reaction at 4 ° C.
  • the cells were reacted for 30 minutes with a goat anti-Peacock IgG antibody conjugated with biotin, and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 g / ml 0.006% and diaminobenzidine H 2 0 2 50 negation ol / L Tris-HCl (P H 7.6) containing buffer coloration said the I Ri Peruokishidaze activity to react for 10 minutes in type X sPLA 2 in the tissue specimens were visualized. In addition, cell nuclei were counterstained by reaction with 0.4% hematoxylin aqueous solution.
  • X-type sPLA 2 positive signal was detected as deposits dark brown di ⁇ amino benzylidene den.
  • Neutralization experiments X-type sPLA 2 signal, prior to the addition of antibody to slide was performed purified X-type sPLA 2 protein and (60 jg / mL) by reacting for 2 hours.
  • Example 16 Cloning of Gene Encoding Mouse X-Type sPLA;
  • two types of primers were designed based on the human X-type sPU ⁇ sequence, and PCR was performed using mouse thymus cDNA as type III.
  • PCR was performed for 40 cycles at 92 ° (: 1 minute, 58 ° 0 for 1 minute, and 72 ° C for 1 minute).
  • mice spleen marathon-ready cDNA (Clontech)
  • the 3'- and 5'-ends were cloned by the rapid amplification of the cDNA ends (RACE) method, and finally the following primers were used.
  • RACE rapid amplification of the cDNA ends
  • mouse type X sPLA 2 is, Valentin et al (J. Biol. Chem., 274 , 44, 31195-31202 (1999)) so as to report, 1 5 consists of one amino acid (SEQ ID NO: 1 4), having a human type X sPLA 2 with respect to 7 0.9% homology was estimated.
  • Mouse X-type sPLA ⁇ has a 28-amino acid pre-peptide at the N-terminal like human X-type sPLA ⁇ ,
  • mouse type X sPLA 2 is different from the human type X sPLA 2, N - linked glycosylation sites had no.
  • Example 17 Enzyme activity of mouse pro-type and mature type-X sPLA
  • the enzymatic activity of the purified pro-type and mature type-X sPLA ! was measured using l-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC) as a substrate.
  • PLPC l-palmitoyl-2-linoleoyl-phosphatidylcholine
  • a 'as shown in FIG. 2 the proform X type sPLA 2 is has failed not only has only 8% of the activity compared to mature.
  • the presence of Arg at the C-terminus of the propeptide suggested that trypsin-like end protease may be involved in cleavage. Therefore, the pro-type X sPLAj was digested with trypsin and analyzed by SDS-PAGE.
  • X-type sPLAi peptide was synthesized by a peptide synthesizer based on the amino acid sequence information taught by the present invention.
  • the obtained peptide was combined with bovine thyrologous purine as an immunogen, and immunized with egret to prepare an antiserum.
  • the IgG fraction was purified using a HiTrap plug-in G-column (Amersham Pharmacia Biotech). The specificity and titer of the antiserum and the purified antibody were measured by the following ELISA method.
  • Bovine serum Al Bumi ting coupled 9 6 ⁇ El plates coated with type X sPLA 3 ⁇ 4 propeptide was (Nunc, Immtmo Maxi Sorp plates) was prepared, after Purodzuku the nonspecific adsorption in 1% bovine serum albumin solution, It was reacted by adding Kohi preparative X type sPLA 2 propenyl peptide antibodies. After washing with PBS (Phosphate-buffered saline, pH 7.4) containing 0.05% Tween20, it was further reacted with peroxidase-conjugated goat anti-Peagle IgG antibody (Cappel), and developed with TBMplus (DAKO). . The coloring reaction was terminated by adding 0.5 mol / L sulfuric acid, and the coloring intensity was measured at a wavelength of 450 nm.
  • PBS Phosphate-buffered saline, pH 7.4
  • Tween20 peroxidase-conjugated goat anti
  • anti-X type sPLA 2 propenyl peptide obtained by the antibody does not react with pro-X-type sPLA 2 or active type X sPLA 3 in EL I SA system shown in Example 3, Purobe peptide was the only one who recognized.
  • peptides were measured by the following ELISA method 1 method. The peptide was reacted with EZ-Link Sulfo-NHS-LC-LC-Biotin (Pierce) and separated by reversed-phase HPLC to prepare a biotinylated peptide.
  • a 96-well plate coated with avidin was prepared, nonspecific adsorption was blocked with a 1% bovine serum albumin solution, and then a biotinylated propeptide was added and reacted for 2 hours. After washing with PBS containing 0.05% Tween20 (Phosphate-buffered saline, pH 7.4), Added type X sPLA 2 propenyl peptide antibody analyte or standard solution to be measured simultaneously, and allowed to react for 1 8 hours. After washing, the plate was further reacted with a peroxidase-conjugated goat anti-Peacock IgG antibody (Cappel), and after washing, TBMplus (DAKO) was added to develop color.
  • PBS Phosphate-buffered saline, pH 7.4
  • Tween20 Phosphate-buffered saline, pH 7.4
  • the coloring reaction was terminated by adding 1N sulfuric acid, and the coloring intensity was measured at a wavelength of 450 nm.
  • Absorbance B obtained with Ong / mL standard solution B "Calculate the percentage of absorbance B obtained with the standard solution at each concentration, and create a standard curve (Fig. 3), which can be used to obtain the unknown sample.
  • the concentration of the peptide contained in the unknown sample was calculated from B / B% obtained from the absorbance.
  • Propeptide was also measured by the following ELISA second method.
  • an anti-human X type sPLA2 peptide antibody was used.
  • Specimens of colorectal tissues of healthy adults and colorectal cancer tissues from colorectal cancer patients were purchased from Biochaiii Inc. (San Leandro, CA). Slide of tissue section, remove the paraffin Nwadzukusu, after removing the endogenous Peruokishi evening one peptidase allowed to react for 30 minutes in methanol containing 0.3% H 2 0 2, was treated with 5% normal turbocharger formic serum for 20 minutes.
  • the cells were reacted with an anti-human X-type sPLA 2 ( ⁇ sagi) propeptide antibody (24 g / mL) in a PBS aqueous solution containing 0.1% bovine serum albumin for 14 hours at 4 ° C. 0.1% Polyoxyethyleiie (20) Washed with PBS aqueous solution containing sorlDitan monolaurate (Tween20; Wako Pure Chemical Industries, Ltd. Osaka), reacted with goat anti-Peacock IgG antibody conjugated with pyotin for 30 minutes, Striped Avidin Reagent Containing Idose (DAKO Corporation, Carpinteria, CA) for 15 minutes.
  • an anti-human X-type sPLA 2 ( ⁇ sagi) propeptide antibody 24 g / mL
  • PBS aqueous solution containing 0.1% bovine serum albumin for 14 hours at 4 ° C. 0.1%
  • a biotin-labeled tylamide solution (DAKO Corporation, Carpinteria, CA) was allowed to react for 15 minutes, followed by thorough washing with a PBS aqueous solution containing 0.1% Tween20. Further, the peroxidase-labeled streptavidin solution was treated for 15 minutes, and similarly washed sufficiently with a 0.1% Tween20-containing PBS aqueous solution. After washing, the peroxidase activity was developed by reacting for 10 minutes in a 50 mmol / L Tris-HCl (pH 7.6) buffer containing 200 ⁇ g / mL diaminobenzidine and 0.006% H2O2.
  • sPLA 2 propeptide type sPLA 2 propeptide were visualized. In addition, cell nuclei were counterstained by reaction with a 0.4% aqueous solution of methoxylin. X-type sPLA 2 propeptide positive signal was detected as a brown deposits di Aminobenjiden. The absorption neutralization experiment of the propeptide positive signal was carried out by reacting with propeptide-conjugated bovine serum albumin (300 ⁇ g /: mL) for 2 hours before adding the antibody to the slide.
  • antibodies specifically recognizing a part of the X-type sPLA 2; Measurement method of active X-type S PLA 2 using the antibody; such as diagnostic kits comprising the antibody is provided .

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Neurosurgery (AREA)
  • Hematology (AREA)
  • Neurology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Psychiatry (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une méthode de dosage immunologique pour sPLA2 de type X. L'utilisation d'un anticorps reconnaissant de façon spécifique une partie de sPLA2 de type X, permet d'obtenir un dosage de sPLA2 de type X activée.
PCT/JP2000/008198 2000-05-24 2000-11-21 Methode de dosage immunologique pour phospholypase a2 de type x Ceased WO2001090195A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU2001214187A AU2001214187A1 (en) 2000-05-24 2000-11-21 Immunoassay method for x-type phospholipase a2
PCT/JP2001/004267 WO2001090196A1 (fr) 2000-05-24 2001-05-22 Procede d'immunodosage d'une phospholipase a2 de type x
AU58798/01A AU5879801A (en) 2000-05-24 2001-05-22 Immunoassay method for X-type phospholipase A2
JP2001587007A JP4726031B2 (ja) 2000-05-24 2001-05-22 Ｘ型ホスホリパーゼａ２の免疫測定方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2000-152967 2000-05-24
JP2000152967 2000-05-24

Publications (1)

Publication Number Publication Date
WO2001090195A1 true WO2001090195A1 (fr) 2001-11-29

Family

ID=18658306

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2000/008198 Ceased WO2001090195A1 (fr) 2000-05-24 2000-11-21 Methode de dosage immunologique pour phospholypase a2 de type x

Country Status (3)

Country Link
JP (1) JP4726031B2 (fr)
AU (1) AU2001214187A1 (fr)
WO (1) WO2001090195A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999020651A1 (fr) * 1997-10-21 1999-04-29 Sankyo Company, Limited Nouveaux composes antifongiques

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6103469A (en) * 1997-11-07 2000-08-15 Incyte Pharmaceuticals, Inc. Human phospholipase A2 protein

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999020651A1 (fr) * 1997-10-21 1999-04-29 Sankyo Company, Limited Nouveaux composes antifongiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CUPILLARD L. ET AL.: "Cloning, chromosomal mapping and expression of a novel human secretory phospholipase A2", J. BIOL. CHEM., vol. 272, no. 25, June 1997 (1997-06-01), pages 15745 - 15752, XP002936702 *
HANASAKI K., ONO T. ET AL.: "Purified group X secretory phospholipase A(2) induced prominent release of arachidonic acid from human myeloid leukemia cells", J. BIOL. CHEM., vol. 274, no. 48, November 1999 (1999-11-01), pages 34203 - 34211, XP002936701 *

Also Published As

Publication number Publication date
JP4726031B2 (ja) 2011-07-20
AU2001214187A1 (en) 2001-12-03

Similar Documents

Publication Publication Date Title
US8673593B2 (en) Antibodies to alpha-synuclein
AU752642B2 (en) Antibody against LAR phosphatase subunit
JP2824150B2 (ja) スペクトリン又はその崩壊生成物の分析による細胞壊死検出
JPWO2005038457A1 (ja) 多量体アディポネクチンの分別測定方法
Weivoda et al. ELISA for human serum leucine-rich alpha-2-glycoprotein-1 employing cytochrome c as the capturing ligand
Nagasaka et al. Defining the pathogenic involvement of desmoglein 4 in pemphigus and staphylococcal scalded skin syndrome
WO2007021255A1 (fr) Anticorps de l’alpha-synucléine
WO2002074805A1 (fr) Protéine rage soluble
WO2013035799A1 (fr) Anticorps apte à se lier à une région spécifique de la périostine, et procédé de mesure de la périostine à l'aide de celui-ci
KR101154550B1 (ko) 폰 빌레브란트 인자 특이적 절단 효소에 대한 항체 및그것을 이용한 분석계
JP2019510210A (ja) 線維症の組み合わせたバイオマーカー測定
KR100884487B1 (ko) 인자 ⅶ-활성화 프로테아제의 돌연변이체 및 특이적 항체를 포함하는 진단학적 조성물
JP5252339B2 (ja) Ｐａｄ４及び抗ｐａｄ４抗体の測定方法並びに関節リウマチの検出方法
Cox et al. Determination of cathepsin S abundance and activity in human plasma and implications for clinical investigation
Eydoux et al. Human pancreatic lipase-related protein 2: tissular localization along the digestive tract and quantification in pancreatic juice using a specific ELISA
JP6389168B2 (ja) 病的な軟骨代謝回転の決定
WO2007074864A1 (fr) Anticorps capable de reconnaitre la dimethylarginine asymetrique, procede pour la production de l'anticorps, et procede pour la detection de proteine ayant un acide amine modifie de maniere post-traductionnelle
WO2001090195A1 (fr) Methode de dosage immunologique pour phospholypase a2 de type x
CN117164715A (zh) 一种抗人激肽释放酶1单克隆抗体及其应用
WO2001090196A1 (fr) Procede d'immunodosage d'une phospholipase a2 de type x
EP2918600B1 (fr) Nouveau peptide et son utilisation
JP2008526937A (ja) ファクターＸＩＩａのフォーム
US20180086844A1 (en) Antibodies against mammal secreted klotho protein
JP5852433B2 (ja) ソルチリンによる動脈硬化の判定方法
JPWO2001090196A1 (ja) Ｘ型ホスホリパーゼａ２の免疫測定方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP