WO2001090196A1 - Procede d'immunodosage d'une phospholipase a2 de type x - Google Patents

Procede d'immunodosage d'une phospholipase a2 de type x Download PDF

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WO2001090196A1
WO2001090196A1 PCT/JP2001/004267 JP0104267W WO0190196A1 WO 2001090196 A1 WO2001090196 A1 WO 2001090196A1 JP 0104267 W JP0104267 W JP 0104267W WO 0190196 A1 WO0190196 A1 WO 0190196A1
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type
spla
cancer
antibody
amino acid
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PCT/JP2001/004267
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English (en)
Japanese (ja)
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Kohji Hanasaki
Keiichi Imagawa
Keiichi Masuta
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Shionogi & Co., Ltd.
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Priority claimed from PCT/JP2000/008198 external-priority patent/WO2001090195A1/fr
Application filed by Shionogi & Co., Ltd. filed Critical Shionogi & Co., Ltd.
Priority to JP2001587007A priority Critical patent/JP4726031B2/ja
Priority to AU58798/01A priority patent/AU5879801A/en
Publication of WO2001090196A1 publication Critical patent/WO2001090196A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • Immunoassay art X phospholipase eight 2 - present invention, X phospholipase A 2 (X-type sPLA 2) which specifically recognizes a part of the antibody; active type X sPLA 2 with antibody A diagnostic kit comprising the antibody.
  • Background art X phospholipase eight 2 - present invention, X phospholipase A 2 (X-type sPLA 2) which specifically recognizes a part of the antibody; active type X sPLA 2 with antibody A diagnostic kit comprising the antibody.
  • Phospholipase 2 (PLA 2 ; EC 3.1.1.4) is a general term for phospholipidases that hydrolyze the 2-acyl ester bond of 3-S / J-phosphodariseride.
  • PLA 2 is involved in the digestion of phospholipids in food, the generation and metabolism of biological membrane phospholipids, and the production of lipid media such as prostaglandin, leukotriene, platelet activating factor (PAF), and lysophospholipid. Functions as an acid cascade initiating enzyme.
  • PLA 2 In mammals are clearly the presence of various PLA 2 summer, its localization, calcium ion (Ca 2+) requirement, based on the substrate specificity, secreted PLA 2, cytosolic PLA 2, Ca 2 ⁇ Independent PLA 2 , and: have been classified into four families of PAF-acetylhydrolase (Balsinde et al., J. Biol. Chem. 272, 16069-16072 (1997)). Of these, the secreted PLA 2 (sPLA 2 ) family is a group of low molecular weight (13,000 to 15,000) PLA 2 molecules secreted extracellularly. In humans, IB, ⁇ , IID, Six types, IIE, V, and X, are known.
  • Human type X sPLA 2 on the basis of the sPLA 2 related sequences from DNA databases were Ri by fetal lung tissue black-learning (Cupillard, et, J. Biol Chem, 272, 15745-15752 (1997)..): Human gene X type sPLA 2 is located in the first 6 on chromosome unlike other sPLA 2.
  • This sPLA 2 has 16 cysteine residues in the molecule, and all of the characteristic intramolecular disulfide bridges present in each of IB type sPLA 2 and IIA / IID / IIE type 51 ) 1 ⁇ 2.
  • having a unique force Rupokishiru group terminal extension structure IIA / IID / IIE type sPLA 2.
  • the raw physical type X sPLA 2 is unknown detail with pathological features, the localization of the expression site, may present enzyme is involved in the regulation of the immune system and inflammatory conditions Presumed.
  • An object of the present invention a part of X-type sPLA 2 specifically recognizes bowtie; to provide a diagnostic kit comprising said antibody; Measurement method of active type X sPLA 2 with antibody .
  • the present inventors have been conducted extensive study with the aim to clarify the physiological function of human type X sPLA 2, in the process, the X-type sPLA 2, the non-active form that has propeptide sequence N- terminus And two active species, the pro-type of which is generated by cleavage of the pro-peptide portion Thus, the amino acid sequence of the propeptide portion cleaved during the activation process was identified.
  • a therapeutic agent for a PLA2-related disease comprising the antibody according to (3) or (6);
  • a cancer diagnostic kit comprising a reagent capable of detecting the polypeptide of (8) or (9) above;
  • kits for diagnosing Alzheimer's disease comprising a reagent capable of detecting the polypeptide according to (8) or (9) above;
  • a kit for diagnosing cirrhosis comprising a reagent capable of detecting the polypeptide according to (8) or (9) above;
  • (22) a method for screening for cancer, Alzheimer's disease or cirrhosis, characterized by detecting the polypeptide according to (8) or (9);
  • X-type sPLA 2 includes prebub-type X-type sPLA 2 and pro-type X-type sPLA 2 And active X-type sPLA 2 (also referred to as mature X-type sPLA 2 ).
  • X-type sPLA 2 is produced as Purebu port type having a signal peptide.
  • the Purebu port type X type sPLA 2 in the amino acid sequence set forth in SEQ ID NO: 3, refers to a polypeptide consisting of amino acids up to Asp 1 2 3 position from a 3 2-position of Me t.
  • the signal peptide is a polypeptide consisting of amino acids from Mei at position 132 to Gly at position 12 in the amino acid sequence of SEQ ID NO: 3.
  • Proform; X-type sPLA 2 occurs connexion by that signal peptide from Purebu port type is disconnected.
  • the proform type X sPLA 2 in the amino acid sequence set forth in SEQ ID NO: 3 means a polypeptide consisting of amino acids up to Asp 1 2 3 position from Gl u one 1 first.
  • pro-type X-type sPLA 2 is the amino acid sequence set forth in SEQ ID NO: 3, is cleaved between one 1-position of Arg and position 1 Gly, in the amino acid sequence according to the propeptide (SEQ ID NO: 3, one 11 A polypeptide consisting of 11 amino acids from Glu at position 1 to Arg at position 11; and active X-type sPLA 2 (in the amino acid sequence of SEQ ID NO: 3, position G to position 1 2 3 Of Asp).
  • this active type X sPLA 2 is it is thought to have a function as Hosuhoripa Ichize A 2.
  • a fragment consisting of a signal peptide and a propeptide may be referred to as a prebub peptide.
  • the Prebu-mouth peptide means a polypeptide consisting of the amino acids from the 132nd position Met to the 11th position Arg in the amino acid sequence of SEQ ID NO: 3 (FIG. 1).
  • a part of a polypeptide consisting of an amino acid sequence from Glu at position 11 to Asp at position 123 in the amino acid sequence of SEQ ID NO: 3 refers to pro-form X-type sPLA 2 Means part of. Preferably, that means the pro-peptide or active type X sPLA 2.
  • the "polypeptide according to (6) or (7)” means Purobe petit de or active type X sPLA 2. The present invention aims to measure the active type X sPLA 2 by immunological means.
  • pro-type X sPLA 2 produces a pair of pro-peptides with active X-type sPLA 2 .
  • the present inventors have found that by specifically measuring Purobe peptide that is produced equimolar mass and active type X sPLA 2 in the course of active type X sPLA 2 or activation, of the active X-type sPLA 2 The measurement method was completed.
  • an antibody that specifically recognizes a polypeptide consisting of amino acids from Glu at position 11 to Arg at position 11 in the amino acid sequence of SEQ ID NO: 3 Means an antibody that recognizes By “specifically sure to identify the propeptide", although the reaction propeptide means cross-reactivity with active type X sPLA 2 is 5% or less'.
  • Poribe peptide specifically recognize antibodies consisting of amino acids from position 1 Gl y up 1 2 3 position of the Asp
  • the active type X sPLA 2 Means an antibody that specifically recognizes.
  • the active X-type s! As specifically recognizes monochromator opening one monoclonal antibody to »LA 2, National Institute of Advanced Industrial Science and Technology Institute of Patent Organism Depositary center one (Japan, Tsukuba, Ibaraki, ⁇ 1-chome 1 address 1 Chuo No. 6) exemplifies a monoclonal antibody produced by hybridoma 3C10 (accession number: FERMBP-7393) deposited on December 12, 2000.
  • the propeptide or active type X sPLA 2 is used to examine the specificity of these antibodies can be used as "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.
  • Secreted PLA 2 is a fatty acid derived from phospholipids found in cell membrane lipoproteins. (Eg, arachidonic acid). Excessive release of fatty acids and the production of various lipid mediators due to their metabolism cause diseases such as septic shock, adult respiratory distress syndrome, tengitis, trauma, bronchial asthma, allergic rhinitis, and rheumatoid arthritis.
  • PLA 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 active 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, colorectal 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, kidney tissue of kidney cancer patients, gallbladder Gallbladder tissue of cancer patients, prostate tissue of prostate cancer patients, lignum tissue of kidney cancer patients, testicular tissue of testicular cancer patients, ovarian tissue of ovarian cancer patients, skin tissue of skin cancer patients, cerebral tissue of Alzheimer's disease patients and in cirrhotic patients with liver tissue, it was confirmed that the X-type sPLA 2 in comparison to their tissue of a normal human is significantly high expression.
  • sPLA a second substrate to the antibody of the re phospholipids or the present invention using as a "reagent", by confirming the activity or expression of type X sPLA 2, cancer, and can be diagnosed Alzheimer's disease or cirrhosis Become.
  • 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, and a western blotting method.
  • a sedimentation reaction method For example, an appropriately diluted patient serum is reacted with an antibody on a microplate on which a solid phase is immobilized, and after washing, an anti-Egret IgG antibody labeled with a secondary antibody, peroxidase, is added. After that, the peroxidase substrate is added to develop a color, and the absorbance at 450 nm is measured, whereby the polypeptide of the present invention can be detected.
  • the kit of the present invention includes, as necessary, various reagents such as a coloring reagent, a reaction stopping reagent, a standard antigen reagent, and a sample pretreatment reagent 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 in addition to the antibody of the present invention.
  • An appropriate reagent according to the measurement method can be appropriately selected and can be attached to the kit of the present invention.
  • cancer particularly colon cancer, lung cancer, liver cancer, stomach cancer, kidney cancer, gallbladder cancer, prostate cancer, Tengler cancer, testicular cancer, ovarian cancer or skin cancer, Group screening for early detection of Alzheimer's disease or cirrhosis is possible.
  • the subject of the biological sample e.g., blood as a sample, by the like diagnostic kits of the present invention, cancers involving active type X s PLA 2, the onset of Aruhha one more disease or cirrhosis
  • the present invention also provides a "screening method" for prediagnosis. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 is a diagram showing the relationship between a prebub-type X-type sPLA 2 , a pro-type X-type sPLA 2 and an active-type X-type sPLA 2 .
  • FIG. 2 is a diagram showing the enzymatic activities of mouse activated X-type sPLA 2 , mouse pro-X-type sPLA 2, and trypsin-enhanced pro-X-type sPLA 2 .
  • M, P and P + T, respectively active means proform and proform type X sPLA 2 was digested with trypsin.
  • Figure 3 shows a standard curve in the ELISA method using the anti-X type sPLA 2 propenyl peptide antibodies of the present invention.
  • Figure 4 is a diagram showing the specificity of the active type X sPLA 2 monoclonal antibody (3 C 10) of the present invention.
  • FIG. 5 is a diagram showing the specificity of the propeptide monoclonal antibody (HPX-6E1) of the present invention.
  • FIG. 6 is a diagram showing a standard curve and specificity of EL I SA system using the active type X sPLA 2 monoclonal antibody of the present invention (3C 10).
  • FIG. 7 is a diagram showing a standard curve and specificity of an ELISA system using the propeptide monoclonal antibody (HPX-6E1) of the present invention.
  • FIG. 8 is a diagram showing measurement results of a sample derived from a colorectal cancer patient using various measurement systems.
  • the present invention relates mainly measurement of active type X sPLA 2 with Purobe peptide.
  • DNA encoding the type X sPLA 2 may be readily manufactured and obtained by a general genetic engineering technique based on the sequence information taught by the present invention (Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989 ) Etc.). Specifically from an appropriate origin which X type sPLA 2 is expressed, a cDNA library was prepared according to a conventional method, using a suitable probe or antibody specific from the stripe library one of the nucleotide sequence of X-type sPLA 2 It can be carried out by selecting a desired clone (see Proc. Natl. Acad. Sci., USA., 78, 6613 (1981); Science, 22, 778 (1983), etc.).
  • cDNA libraries are commercially available, and in the present invention, such cDNA libraries, for example, various cDNA libraries commercially available from Clontech can also be used.
  • Type X sPLA 2 of DNA a method of subscription-learning from a cDNA library one can follow particularly limited Sarezu the usual way.
  • the professional one blanking to be used herein is DNA chemically synthesized based on information about the sequence of the X-type sPLA 2, etc. can be generally exemplified, even better of course already present invention DNA itself or fragments obtained It can be used for
  • sense primers and antisense primers set based on the nucleotide sequence information of the DNA of the present invention can also be used as screening probes.
  • DNA encoding a type X sPLA 2 is conventional chemical methods, for example tricalcium E ester method (Narang et al., Meth. Enzymol., 68, 90-108 (1979)) or phosphate Wells ester It can be synthesized by the method (Brown et al "Meth. Enzymol” 68, 109-151 (1979)).
  • X-type sPLA 2 protein according to the base sequence information of the X-type sPLA 2, genetic engineering proposed method (Sc i enc e, 224, 1431 (1984);.... Bi ochem Bi ophys Res Comm, 1 30, 692 Natl. Accad. ScI., USA., 80, 5990 (1983), etc.) can be obtained as a recombinant protein. 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 yeast.
  • Examples of the vertebrate cells include COS cells (Cell, 23, 175 (1981)) which are monkey cells and Chinese vertebrate cells. ⁇ Hamster ovary cells are often used.
  • an expression vector one having a promoter located at the upstream of the gene to be expressed usually, one having an RNA splice site, polyadenylation site, transcription termination sequence, etc. can be used. It may have a replication origin.
  • the expression vector include, for example, pSV2dhfr (Mol. Cell. Biol., 1, 854 (1981)), which has an initial promoter of SV40.
  • yeasts are generally used, and among them, Saccharomyces yeasts can be used.
  • yeast expression vector for example, pAM82 (Pro Natl. Acad. Sci., USA., 80, 1 (1983)) having an acid phosphatase gene promoter can be used.
  • Escherichia coli and Bacillus subtilis are commonly used as prokaryotic hosts.
  • a plasmid vector capable of replicating in the host bacterium is used, and a promoter and SD sequence upstream of the gene so that the desired gene can be expressed in the vector, Further, it is preferable to use an expression plasmid having an initiation codon required for initiation of protein synthesis.
  • E. coli K12 strain or the like is used.
  • PBR322 and its improved vectors 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 promoter and the like can be used.
  • 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.
  • pSVL SV40 Late Promo One Night Of the human type X sPLA vector one containing two genes of the present invention downstream, to create a transformant by introducing into monkey-derived cells C0S-7, the transformants 5% C02 presence, 37 ° by culturing for 3 days in C, and recombinant human type X sPLA 2 protein that obtained 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 include various types of liquid chromatography, dialysis, and combinations thereof.
  • Purified X-type sPLA 2 is for containing a proform type X sPLA 2, (if example embodiment, Supuchirishin like endoproteases, trypsin, etc.) Suitable proteases in the child cut by, obtaining active type X sPLA 2 Can be done.
  • X-type sPLA 2 protein or X-type sPLA 2 protein fragment (e.g. Purobe Petit de), based on the amino acid sequence information taught by the present invention, can be synthesized by a peptide synthesizer. Preparation of antibodies against the protein of the present invention
  • An antibody against the protein of the present invention is produced by the following method.
  • Lymphocytes are extracted from the spleen or lymph node of a mouse rat immunized with the above-mentioned immunogen, fused with myeloid cells, and Kohler and Milstein's method (Nature, 256, 495-497 (1975))
  • a monoclonal antibody can be produced from the hybridoma.
  • a monoclonal antibody against the Purobe peptide or active type X sPLA 2 by the following steps:
  • the present invention with reference to ⁇ for Purobe peptide or active type X sPLA 2, it is possible to perform measurement of active type X sPLA 2 protein.
  • the measurement method of the present invention The amount of the body, antigen, or antibody-antigen complex corresponding to the amount of antigen (propeptide or active X-type sPLA 2 ) in the measurement solution is detected by chemical or physical means, and the amount is detected. Any measurement method may be used as long as it is a measurement method calculated from a standard curve prepared using a standard solution containing the 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, m I,, as the "C etc. are used. Enzymes, Peruokishidaze,
  • CDNA encoding human type X sPLA 2 is a human placental cDNA as ⁇ obtain Ri by the PCR method. The following primers were used for PCR.
  • hGX-S 5'-ctgtgtacgcgtccatgctgctcctgctactgccgtc-3 '(SEQ ID NO: 5)
  • hGX-AS 5'-tcaa tgcggccgctcagtcacact tgggcgcgtcc-3 (SEQ ID NO: 6)
  • hGGX-S has a Kozak sequence and a Mlul recognition site.
  • HGX-AS also has a NoU recognition site.
  • PCR was performed for 35 cycles under the conditions of 30 degrees at 94 degrees, 50 seconds at 50 degrees, and 2 minutes at 72 degrees.
  • the PCR amplified fragment was digested with Mlul and Notl, and introduced into pBS-SK (-).
  • force Rupokishiru group terminal to the six X-type by adding the His residue sPLA 2 -HisTag body, HGx - S-flop la y M a ⁇ beauty hGX-H6AS flops la y M a (5 ' - It was constructed by PCR using tcaagtgcggccgctcaatggtg tggtgatgatggtcacaci tgggcgagtccggc t-3 ': system!
  • PCR amplified fragment was cut with NGU and Xhol, and replaced with the corresponding region of the X-type sPLA 2 plasmid. After confirming the sequence of the region amplified by PCR, the cDNA was inserted downstream of the SR- ⁇ promoter in an expression vector for mammalian cells.
  • Example 2 Expression, purification and characterization of human X-type sPLA ⁇ -HisTag recombinant protein
  • LipofectAMINE reagent (Life Technologies, Inc.) was added to 293 cells derived from human embryonic kidney (293T cells) transformed with SV-40 at 0.5 / zg of hygromycinB resistance gene together with 20 g of plasmid containing sPLA 2 -HisTag cDNA of type X. ). 293T clones stably expressing X-type sPLA 2 -HisTag were obtained by selection based on their resistance to hygromycin B. Expression of the recombinant protein was detected by a chromogenic PLA 2 activity assay.
  • a HisTag protein 500 mmol / L imidazole, the 20 mmol / L sodium phosphate buffer containing 0.5 mol / L sodium chloride (pH 7.4)
  • the fraction containing the obtained X-type sPLA 2 -HisTag protein was dialyzed against a 20 mmol / L Tris-HCl buffer containing 1 mmol / L phenylmethyl sulfonic acid, and a HiTrap Q column (Amersham Pharmacia Biotech). ) to was added.
  • X-type sPLA 2 - cut of the band of HisTag protein the amino acid sequence of its N-terminal was analyzed by Applied Biosystems Procise Sequencer.
  • X-type sPLA 2 of purified protein preparation obtained by adding a His residue at the C-terminal obtained from the above had a PLA 2 activity.
  • SDS-PAGE analysis most were molecular species with a molecular weight of 16-kDa, but some showed a molecular weight of 14-kDa.
  • the antiserum was prepared and the IgG fraction HiTrap Protein G - force ram (Amersham Pharmacia Biotech) Purified using The specificity and titer of the antiserum and the purified antibody were measured by the following ELISA method.
  • the plate After washing with PBS containing 0.05% Tween20 (Phosphate-buffered saline, H 7.4), the plate is further reacted with a peroxidase-conjugated anti-egg igG antibody (Immunotech), and finally 0.5 fflg / mL of 0-phenylenediamine and 0.013 ⁇ 4 (v / v) was H 2 0 50 comprising 2 mmo L / L click E phosphate sodium buffer is added (pH 5.3) and developed in. The color reaction was terminated by adding 2 mol / L sulfuric acid, and the color intensity was measured at a wavelength of 492 nm.
  • the plate type X sPLA 2 - coated with HisTag or Ax Bok Hi sTag protein was analyzed by method towards the above.
  • anti-X type sPLA 2 antibody thus obtained contained human type X sPLA 2 was added HisTag although to recognize, did not react with type IB and type IIA sPLA 2. Furthermore, antibodies prepared did not react with HisTag additional proteins not related to the X-type sPLA 2. Therefore, HisTag additional part, it became clear that not Epitopu anti X type sPLA 2 antibody.
  • the CH0 cells that the enzyme was stable expression was prepared as described above.
  • the CH0 cells were cultured in Dulbecco's Modified Idal medium containing 10% fetal calf serum and, when the cells became confluent, changed to serum-free PM-1000 medium (Eiken, Japan). Was further cultured for 3 days.
  • the resulting culture supernatant was added to Afi two tee one column coupled with the anti-type X sI> LA 2 antibodies.
  • This column is an anti-type X sPLA 2 IgG (30 mg), HiTrap N- hydroxysuccinimide imide activated column according to a conventional method; prepared by coupling the (5 mL Amersham Pharmacia Biotech). After washing the column with sodium phosphate buffer (pH 7.4), the bound proteins were eluted with 0.1 mol / L glycine-HCl (H1.7). This eluted fraction was added to the above-mentioned reversed-phase HPLC column, and separated with a concentration gradient of acetonitrile in 0.1% trifluoroacetic acid from 20 to 40 ⁇ i.
  • N-glycosidase F treatment By N-glycosidase F treatment, it was aggregated into two molecular species (14-kDa and 13-kDa), and as a result of their N-terminal amino acid sequence analysis, the majority of the 13-kDa protein (GILELAGT: SEQ ID NO: 8) was estimation mature human type X sPLA 2 (in the amino acid sequence set forth in SEQ ID NO: 3, a polypeptide consisting of 1 2 3 position of Asp from position 1 Gly) is, on the other hand, proteins of a few 14-kDa is peptide consisting of 11 residues at the N-terminus of the active X-type sPLA 2: in the amino acid sequence set forth in (EASRILRVHRR SEQ ID NO: 9) is added professional type (SEQ ID NO: 3, 1 2 3 position from a 1 1-position of Glu Asp polypeptide). Therefore, it shows a wide range of molecular weights over 15-18-kDa
  • the substrate specificity of purified human type X sPLA 2 each purified sample, position to have Parumichi phosphate, various different fatty acids were purchased commercially having different 1 two phosphorus lipid polar group with the 2-position They were tested as substrates.
  • Each sPLA 2 protein for enzymatic activity (IB type, I IA type sPLA 2 ⁇ Pi human type X sPLA 2 in HPLC 3 fractions) from each phospholipid 1 mmo l / L and 3 mmo l / L Dokishikoru acid It was measured under mixed micelle conditions.
  • the present inventors have found that in the process of purification of recombinant human type X sPLA 2 protein, active X-type sPLA 2 propenyl peptide consisting of 11 amino acids at the N-terminus of the protein revealed the presence of additional pro-type X-type sPLA 2, the further substrate specificity analysis, pro enzyme is compared to the activity enzyme The activity was likely to be very low.
  • the SignalP computer analysis to predict the cleavage site of the signal sequence (Nielsen et al., Protein Eng., 12, 3-9 (1999)), the site which is most susceptible to signal peptide cleavage is analyzed.
  • Specimens of healthy adult colon tissue and colon cancer tissue from colorectal cancer patients were purchased from Biochain Inc. (San Leandro, CA). The slide of the tissue section was removed from paraffin wax, reacted with methanol containing 0.3% H 2 O 2 for 30 minutes to remove endogenous peroxidase, and then treated with 5% normal goat serum for 20 minutes. Then, in PBS containing 0.1% bovine serum albumin, anti-X type sPLA 2 antibody (6 ii 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, 50 mmol / L Tris-HCl (H 7.6) were colored with by Ri Peruokishidaze activity to react for 10 minutes in a buffer tissue specimen containing 200 g / mL of Jiamino base Njijin and 0.006 H 2 0 2 X-type sPLA 2 in the inside was 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 g / mL) by reacting for 2 hours.
  • a positive signal is X-type sPLA 2
  • is hardly detected in normal colon tissues was detected strongly in cancer cells of colon adenocarcinoma tissues.
  • these signals it was confirmed to be specific to type X sPLA 2 since disappeared by the addition of X-type sPLA 2 protein.
  • these positive signals were not observed in IgG prepared from non-immunized egrets. From the above results, the expression of type X sPLA 2 protein in colorectal cancer is Rukoto have significantly high expression was suggested.
  • Example 7 Immunohistochemical analysis of human lung tissue using anti-X type sPLA and antibody
  • Specimens of healthy adult lung tissue and lung tissue from lung cancer patients were purchased from Biocha in Inc. (San Leandro, CA). Slide of tissue section, except take paraffin wax, after removing the endogenous Peruokishita Ichize reacted for 30 minutes in methanol containing 0. 33 ⁇ 4 H 2 0 2, treated with 5% normal turbocharger formic serum for 20 minutes did. Then, in PBS with 0. 1 3 ⁇ 4 bovine serum albumin, anti-X type sPLA 2 antibody (6 ng / mL) and 14 hours, the mixture was reacted 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, Peruokishi by reacting for 10 minutes at 200 g / Jiamino base Njijin and 0.006% of mL H 2 0 50 comprising 2 mmo l / L Tr i s- HC l (H 7. 6) buffer and colored to Daze activity type X sPLA 2 in the tissue specimens were visualized. In addition, cell nuclei were counterstained by reaction with a 0. hematoxylin aqueous solution.
  • X-type sPLA 2 positive signal was detected as deposits diaminobenzidine den dark brown.
  • 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 g / mL) by reacting for 2 hours.
  • Specimens of healthy adult liver tissue and lung tissue from liver cancer patients were purchased from Biochain Inc. (San Leandro, CA). Slide of tissue section, except take paraffin wax, after removing the endogenous Peruokishitaze reacted for 30 minutes in methanol containing 0.33 ⁇ 4 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 g / mL) and 14 hours, the mixture was reacted at 4 ° C.
  • X-type sPLA 2 positive signal was detected as deposits diaminobenzidine den dark brown.
  • 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 g / 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 Peruokishita Ichize reacted for 30 minutes in methanol containing 0.33 ⁇ 4 H 2 0 2, was treated with 5% normal turbocharger formic serum for 20 minutes . Then, in PBS with 0.13 ⁇ 4 bovine serum albumin, anti-X type sPLA 2 3 ⁇ 4 body (6 g / mL) and 14 hours, the mixture was reacted at 4 ° C.
  • X-type sPLA 2 positive signal was detected as deposits diaminobenzidine den dark brown.
  • Neutralization experiments X-type sPLA 2 signal, prior to the addition of antibody to slide, was purified X-type sPLA 2 protein and (60 IX g / mL) carried out by reacting for 2 hours.
  • Specimens of healthy adult kidney tissue and lung tissue from kidney cancer patients were purchased from Biochain Inc. (San Leandro, CA). Slide of tissue section, except take paraffin wax, after removing the endogenous Peruokishitaze reacted for 30 minutes in methanol containing 0.33 ⁇ 4 H 2 0 2, was treated with 5% normal turbocharger formic serum for 20 minutes. Thereafter, 0.1%. In PBS containing bovine serum Alp Min, anti X type sPLA 2 antibody (6 ng / mL) and 14 hours, allowed to react at 4 ° C.
  • X-type sPLA 2 positive signal was detected as deposits diaminobenzidine den dark brown.
  • Neutralization experiments X-type sPLA 2 signal, prior to the addition of antibody to slide, was purified X-type sPLA 2 protein and (60 / ig / mL) carried out by reacting for 2 hours.
  • Specimens of healthy adult gallbladder tissue and lung tissue from gallbladder cancer patients were purchased from Biochain Inc. (San Leandro, CA). Slide of tissue section, except take paraffin wax, after removing the 0.3% H 2 0 2 is reacted for 30 minutes in methanol containing endogenous Peruokishi evening Ichize, treated with 5% normal turbocharger formic serum for 20 minutes did. Then, in PBS containing 0.1% bovine serum albumin, anti-X type sPLA 2 antibody (6 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 mmol / L Tris-HCl (pH 7.6) X of by reaction for 10 min in buffer and colored the Peruokishi Daze active tissue specimens containing 200 / mL of Jiamino base Njijin and 0.006% or 0 2 Type sPLA 2 was visualized. Also, 0. «Hematoki Cell nuclei were counterstained by reaction with aqueous syrin.
  • X-type sPLA 2 positive signal was detected as deposits diaminobenzidine den dark brown.
  • 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 iig / mL) by reacting for 2 hours.
  • Specimens of healthy adult prostate tissue and prostate tissue from prostate cancer patients were purchased from Biochain Inc. (San Leandro, CA). Slide of tissue section, remove the Parafinwa' box, after removing the endogenous Peruoki sheet evening over peptidase reacted methanol for 30 minutes containing 0.3% H 2 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, 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). Washing ⁇ , 200 g / Jiamino base Njijin and 0.006% or in mL H 2 0 2 50 mmol / L Tris-HCl (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 g / mL) by reacting for 2 hours.
  • Specimens of healthy adult Teng tissue and Teng tissue from Teng cancer patients were purchased from Biocha in Inc. (San Leandro, CA). Slide of tissue section, remove the paraffin wax, 0. 3% H 2 0 2 after removal of the 3 0 min reacted endogenous Peruokishi evening over zero in methanol containing, 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 or Labo rat or ies).
  • a peroxidase-containing avidin-biotin complex reagent Vector or Labo rat or ies.
  • 200 g / mL 50 mmo l / L Tr is -HC l containing the Jiamino base Njijin a 0. 006% H 2 0 2 in (H 7. 6) Ri by the reacting for 10 minutes in buffer the Peruokishida Ichize X type sPLA 2 activity was colored 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 2 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. (San Leandro, CA). Slide of tissue section, remove the Parafinwa' box, after removal of the 0.3% H 2 0 2 is reacted for 30 minutes in methanol containing endogenous Peruoki Shitaze were treated with 5% normal turbocharger formic serum for 20 minutes. Then, in PBS with 0.13 ⁇ 4 bovine serum albumin, anti-X type sPLA 2 antibody (6 g / mL) and 14 hours, the mixture was reacted at 4 ° C.
  • X-type sPLA 2 positive signal was detected as deposits dark brown di ⁇ amino benzylidene den.
  • X-type, neutralization experiments sPLA 2 signal, prior to the addition of antibody to slide was performed purified X-type sPLA 2 Midorishiro cytoplasm and (60 g / mL) by reacting for 2 hours.
  • a positive signal is X-type sPLA 2
  • senile plaques senile plaque
  • neurofibrillary tangles site neurofibrillary tangle regions
  • Specimens of healthy adult liver tissue and liver tissue from cirrhosis patients were purchased from Biochain Inc. (San Leandro, CA). The slides of the tissue sections were treated with 5 normal goat sera for 20 minutes after removing paraffin wax, 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 albumin, anti-X type sPLA 2 antibody (6 ng / mL) and 14 hours to reaction at 4. After washing with PBS, the cells were reacted for 30 minutes with a heron IgG antibody conjugated with biotin and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories).
  • a positive signal is X-type sPLA 2
  • in normal liver tissues was detected with low levels in the liver lobule and Kup ⁇ ⁇ er stellate cells, in patients with cirrhosis derived from liver tissue, observed significant enhanced expression in sham lobule Was done.
  • These signals it was confirmed to be specific to type X sPLA 2 since disappeared by the addition of X-type sPLA 2 protein.
  • these positive signals were not observed in IgG prepared from non-immunized egret.
  • PCR was performed for 40 cycles under the conditions of 1 minute at 92, 1 minute at 58, and 1 minute at 72 ° C.
  • the 3,-and 5'-ends were cloned by the rapid amplification of the cDNA ends (RACE) method, and finally the following primers were used: Te, the coding region of the mouse type X sPLA 2 cDNA, was obtained by PCR mouse spleen cDNA as ⁇ .
  • mouse type X sPLA 2 is, Valentin ⁇ (J. Biol. Chem ., 274 , 44, 31195-31202 (1999)) so as to report, to have a 1 5 1 consists amino acid (SEQ ID NO: 1 4), 7 0.9% homology to the human X-type sPLA 2 was estimated.
  • Mouse type X sPLA 2 has a pre-pro peptide consisting of 2 8 amino acids in the N-terminal as well as the human type X sPLA 2, having an extended structure in the C-terminus. However, 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 activated X-type sPLA
  • mouse type X sPLA 2 is I line in the same manner as described in Example 4.
  • Purified proform and enzymatic activity of active type X sPLA 2 was determined I- palmitoyl-2- linoleoy Bok phosphatidylcholine a (PLPC) as substrate. As shown in FIG. 2, the proform X type sPLA 2 were not only has only 8% of the activity compared to the active form. The presence of Arg at the C-terminus of the propeptide suggests that trypsin-like endogenous protease may be involved in cleavage.
  • X-type sPLA 2 Purobe peptide is based on the amino acid sequence information taught by the present invention Synthesized with a peptide synthesizer. The resulting probeptide was combined with bovine thyrologous purine as an immunogen, and immunized with egret to prepare 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.
  • a 96-well plate coated with avidin was prepared, nonspecific adsorption was blocked with a 1% bovine serum albumin solution, and then biotinylated probepeptide was added and reacted for 2 hours.
  • PBS containing 0.05% T een20 Phosphate-buffered saline, pH 7.4
  • T een20 Phosphate-buffered saline, pH 7.4
  • the added sample or standard solution to measure the anti-X type sPLA 2 Purobe peptide antibodies were simultaneously allowed to react for 1 8 hours. After washing, it was further reacted with peroxidase-conjugated goat anti-Peacock IgG antibody (Cappel), and after washing, TBMplus (DAK0) was added to develop color.
  • the color reaction was terminated by adding N sulfuric acid, and the color intensity was measured at a wavelength of 450 nm.
  • a standard curve (Fig. 3) was created by calculating the percentage B / B hinder% of the absorbance B obtained with the standard solution at each concentration relative to the absorbance Bo obtained with the standard solution of 0 ng / raL, and used to calculate the unknown sample.
  • the propeptide concentration contained in the unknown sample was calculated from the obtained absorbance, and the propeptide was also measured by the following ELISA method 2.
  • anti-human X-type sPLA2 propeptide derivative was used.
  • Specimens of colorectal tissues of healthy adults and colorectal cancer tissues from colorectal cancer patients were purchased from Biochain Inc. (San Leandro, CA). Slide of tissue section, remove the paraffin wax, after removing the endogenous Peruokishita 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.
  • a biotin-labeled ilamide 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.
  • the peroxidase-labeled streptavidin solution was further treated for 15 minutes, and similarly washed sufficiently with a PBS aqueous solution containing 0.1% Tween20.
  • Splenocytes were prepared from mice with the highest antibody titers, and cell fusion was performed with mouse myeloma cells in the presence of PEG1500.
  • the produced antibody-producing hybridomas were detected by the RIA method described below.
  • Goat anti-mouse IgG was immobilized on a 96-well plate and non-specific adsorption was blocked with PROC-ACE (Dainippon Pharmaceutical), and the monoclonal antibody contained was captured by reacting with the hybridoma culture supernatant. After washing, reacted radioactive iodine-labeled X-type sPLA 2 protein was determined residual after washing radioactivity.
  • the hybridoma was registered as FERMBP-73 993 (accession number) under the Patent Organism Depositary Center for Patented Organisms, National Institute of Advanced Industrial Science and Technology (1-1 Tsukuba East, Ibaraki, Japan 1 1 Central No. 6) has been commissioned.
  • a peptide containing the amino acid sequence at positions ⁇ 11 to 10 in the amino acid sequence described in SEQ ID NO: 3 that had been biotinylated was reacted.
  • the plate was further reacted with peroxidase-labeled streptavidin (Pierce), and finally the color was developed by adding the DAKO TMB One-Step Substrate System (DAKO).
  • DAKO DAKO TMB One-Step Substrate System
  • Splenocytes were prepared from mice with the highest antibody titers, and cell fusion was performed with mouse myeloma cells in the presence of PEG1500.
  • the produced antibody-producing hybridomas were detected by the ELISA method described above.
  • the High Priestess de one Ma detected repeated black one nin grayed by limiting dilution, were established murine monoclonal antibody production High Priestess dormer strain HPX-6E1 reactive to X-type sPLA 2 Purobe peptide.
  • the hybridoma was registered as FERMBP-7 5 4 7 (accession number) under the Patent Organism Depositary Center for Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Japan. 6) Has been commissioned.
  • HPX-6E1 results antibodies of the specificity was examined in ELISA method described above, the decrease in absorbance was not observed in the case where the active added including X-type S PLA 2 proteins, addition of X-type sPLA 2 propeptide was only observed inhibition of binding when present antibody that has been shown to specifically recognize the X-type S PLA 2 propeptide ( Figure 5).
  • the X-type sPLA 2 propenyl peptide-specific monoclonal antibody HPX-6E1 performs mass culture in a serum-free medium, were added directly to the culture supernatant to HiTrap Protein G column, the purified antibodies by elution under the above conditions Obtained.
  • 3C10 antibody cross-linking column and HF-6E1 antibody cross-linking column were prepared.
  • DAKO DAKO TMB One-Step Substrate System
  • HPX-6E 1 Purified antibody was solid phase 96 Uerupure Bok, after blocking nonspecific adsorption in Block Ace (Dainippon Pharmaceutical), as a specimen, active X-type S PLA 2 or proform X type different concentrations reacting the solution containing the sPLA 2, it was captured sPLA 2 contained in the solution. After washing, it was reacted with anti-X type sPLA 2 antibody IgG fraction Piochin of. After washing, the plate was further reacted with peroxidase-labeled streptavidin (Pierce), and finally, a color was developed by adding a DAKO TMB One-Step Substrate System (DAKO), and the color intensity was measured at a wavelength of 450 nm.
  • DAKO DAKO TMB One-Step Substrate System
  • X-type sPLA 2 specific ELISA system Activated using a monoclonal antibody and pro forms: X-type sPLA 2 specific ELISA system and, using a mold nonspecific X type sPLA 2 measurement system using a polyclonal antibody, a normal person serum and colon cancer patient sera the X-type sPLA 2 concentration was determined.
  • the quantitative measurement system was able to detect samples with high levels in colorectal cancer patients. And pairs to this, in a proform type X sPLA 2 specific assay system ⁇ Pi-type non-specific measurement system could not detect a sample value definitive in patients.
  • the grades, active X-type sPLA 2 specific measurement system using the created made the active type X sPLA 2 specific antibodies in the present invention have shown the potential to be clinically useful in the diagnosis of colorectal cancer ( ( Figure 8). Industrial applicability
  • X-type part of sPLA 2 antibodies specifically recognizing; method of measuring the active type X sPLA 2 with said antibody; such as diagnostic kits comprising the antibody is provided.
  • X type sPLA 2 By using the measurement method of the present invention, along with specific becomes possible diseases X type sPLA 2 is involved specifically, certain diseases caused by type X sPLA 2, in particular colon cancer, lung cancer, liver cancer, stomach cancer, A kit for diagnosing kidney cancer, gallbladder cancer, prostate cancer, kidney cancer, testicular cancer, ovarian cancer, skin cancer, Al-Hachi-Ima-ichi disease, and cirrhosis is provided.

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Abstract

L'invention concerne un procédé de dosage d'une sPLA2. L'utilisation d'un anticorps reconnaissant spécifiquement une partie de sPLA2 de type X rend possible le dosage de sPLA2 de type X, activée.
PCT/JP2001/004267 2000-05-24 2001-05-22 Procede d'immunodosage d'une phospholipase a2 de type x WO2001090196A1 (fr)

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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
AU58798/01A AU5879801A (en) 2000-05-24 2001-05-22 Immunoassay method for X-type phospholipase A2

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JP2000-152967 2000-05-24
JP2000152967 2000-05-24
PCT/JP2000/008198 WO2001090195A1 (fr) 2000-05-24 2000-11-21 Methode de dosage immunologique pour phospholypase a2 de type x
JPPCT/JP00/08198 2000-11-21

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003101487A1 (fr) * 2002-05-31 2003-12-11 Mcgill University Utilisation d'inhibiteurs de la phospholipase a2 pour le traitement, la prevention ou le diagnostic des maladies neurales inflammatoires ou demyelinisantes
JP2015506672A (ja) * 2011-12-07 2015-03-05 センター ナショナル デ ラ リシェルシェ サイエンティフィック（シーエヌアールエス） 抗ｓＰＬＡ２−Ｘ抗体およびその使用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999024587A2 (fr) * 1997-11-07 1999-05-20 Incyte Pharmaceuticals, Inc. Proteine de type phospholipase a2 d'origine humaine et adn la codant

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999024587A2 (fr) * 1997-11-07 1999-05-20 Incyte Pharmaceuticals, Inc. Proteine de type phospholipase a2 d'origine humaine et adn la codant

Non-Patent Citations (3)

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Title
BALSINDE J.: "Cloning, chromosomal mapping and expression of a novel human secretory phospholipase A2", CHEMTRACTS, vol. 11, no. 3, 1998, pages 180 - 183, XP002944795 *
CUPILLARD L.: "Cloning, chromosomal mapping and expression of a novel human secretory phospholipase A2", J. BIOL. CHEM., vol. 272, no. 25, 1997, pages 15745 - 15752, XP002944794 *
HANASAKI K.: "Purified group X secretory phospholipase A2 induced prominent release of arachidonic acid human myeloid leukemia cells", J. BIOL. CHEM., vol. 274, no. 48, 1999, pages 34203 - 34211, XP002944796 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003101487A1 (fr) * 2002-05-31 2003-12-11 Mcgill University Utilisation d'inhibiteurs de la phospholipase a2 pour le traitement, la prevention ou le diagnostic des maladies neurales inflammatoires ou demyelinisantes
JP2015506672A (ja) * 2011-12-07 2015-03-05 センター ナショナル デ ラ リシェルシェ サイエンティフィック（シーエヌアールエス） 抗ｓＰＬＡ２−Ｘ抗体およびその使用

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