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KMID : 0352519920290020423
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Korea Univercity Medical Journal
1992 Volume.29 No. 2 p.423 ~ p.432
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A Study on the Production of the Monoclonal Antibody Against
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Abstract
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This study was devised to produce monoclonal antibody against hepatocelullar carcinoma specific Υ-glutamyltranspeptidase(HCC GGT), which proceeded by first purifying the HCC GGY using ion exchange chromatography and immunoaffinity
chromatography.
Subsequently, a mouse immunized with the purified enzyme. and then spleen cells taken from the immunized mouse was fused with mouse myeloma cells(SP2-Ag 14) to form the hybridoma.
The following results were obtained by characterization of the monoclonal antibody of the hybridoma using immunoglobulin isotyping and immunoblot.
1. The purification of HCC GGT by means of ion exchange chromatography and immunoaffinity chromatography produced a yield of 29.41%, a purification fold of 167.87, and final specific activity of 21units per mg of protein.
2. Direct enzyme linked immunoadsorbent assay (ELISA) method was used to measure the antibody level against HCC GGT. An antibody titration of the blood sample taken from the mouse immunized with the above enzyme showed value over 1 : 5, 120
3. The cell fusion of the mouse myeloma cell and the spleen cell of the immunized mouse was generated 14 positive wells in 221 wells. (specific fusion efficacy of 6.33%) Only one IgG2a monoclonal antibody was determined by both repetitive cloning
procedures and immunoglobulin subisotyping.
4. A antigen-antibody cross matching after the western blot revealed a specific reaction between the HCC GGT antigen at the 40kd band and the monoclonal antibody of the hybridoma. And the monoclonal antibody did not react with GGT derived from
normal
liver or kidney.
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