| Project/Area Number |
63570571
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Hematology
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
OKUMA Minoru Kyoto University, Faculty of Medicine, Professor, 医学部, 教授 (50026986)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1989: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1988: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | platelet / collagen receptor / antiplatelet antibody / megakaryocyte / immunoaffinity chromatography / monoclonal antibody / immunoblotting / glycoprotein / アフィニティ-クロマトグラフィ- / モノクロ-ナル抗体 / 血小板凝集 / コラーゲン受容体 / 巨核芽球cell line / CMK |
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| Research Abstract |
1. Characteristics of a putative platelet collagen receptor as studied by the use of antiplatelet antibody from the ITP patient (Y.A.IgG). Platelet membrane protein recognized by Y.A.IGG was demonstrated to have a 62 kD molecular weight by an immunoblot technique. This result supports the concept that this antigen (P62) is a collagen receptor on the platelet. As P62 specifically bound to insoluble collagen (bovine type I), its functional significance was suggested. Aggregation of rabbit platelets induced blv Y.A.IGG implied that P62 is not species specific. As immunologically reactive P62 antigen was detected in about 60% of CMK cells (a human megakaryocytic cell line) by indirect immunofluorescence test, the collagen receptor could be expressed on megakaryocytes.
2. Isolation of P62 by immunoaffinity chromatography. We have tried to purify P62 using Fab fragments of Y.A.IgG as an immobilized ligand for immunoaffinity chromatog raphy. The purification procedures consisted of solubilization of platelet membranes with 1% Triton X-100, affinity chromatographies on columns of Fab-Formylcellulofine and wheat germ agglutinin agarose. Several glycoproteins including P62 were eluted on the wheat germ agglutinin affinity chromatography. Less than 1119 of P62 was recovered from 200mg of platelet membranes prepared from out-dated platelet concentrates. This purification procedures is still so insufficient that other methods including high performance liquid chromatography on preparative SDS-PAGE should be adopted.
3. Monoclonal antibodies obtained by the use of peripheral lymphocytes from the ITP patient with the anti-P62 antibody. We constructed human-human hybridomas by using Epstein-Barr virus-transformed peripheral lymphocytes of the ITP patient with human B lymphoblastoid cell line AC33. One of the monoclonal antibodies produced by the
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