| Analysis of the human monoclonal antibody-defined ovarian cancer antigen epitopePostgraduate: Xiao Bing-Yi Tutor: Wu Su-YunDepartment of pathophysiology, Department of Tumor Immunology Research Jiangxi Medical College, NanchangAbstractMany tumor markers in ovarian cancer have been defined by monoclonal antibodies since the lymphocyte hybridoma technique was established by Kohler and Milstein in 1975. A newmarker, antigen CAT4, was detected by the human monoclonal antibody (HMcAb) againstovarian cancer, designated AT4, which was secreted by the immortalized DNA-transfected cell line AT4 constructed in our laboratory in 1999.In this study HMcAb AT4 purified with caprylic acid-ammonium sulphate precipitation was used as the probe to identify the presence of CAT4. The crude membrane protein extract, which was yielded by the treatment of whole viable human carcinoma cells of ovary with a single-phase solution of 2.5% (vol/vol) 1-butanol, was analyzed by using Dot blotting, SDS- PAGE, Western blotting for the understanding of the molecular characteristics of CAT4 epitope.HMcAb AT4 was purified from spend culture medium of the transfectant cell line AT4. The molecular weights of HMcAb AT4 were mainly 54.3 kD and 25.6 kD in SDS-PAGE.This study showed that CAT4 detected by HMcAb AT4 with Dot blotting could be present in a 2.5% crude 1-butanol extract (OV-CBE) from the surfaces of living human ovarian carcinoma cells. It was determined chemically that CAT4 is a protein, not glycoprotein or glycolipid. CAT4, lost its antigenicity after the treatment with SDS and P -mercaptoethanol. The surface epitope recognized by HMcAb AT4 is a conformation-dependant peptide. The molecular weights of cauiebutanol extract (OV-CBE) were 91.8kDN 88.1kD> 78.0 kD> 76.5 kD- 74.9kD^ 69.MK 59.9kD> 56.4 IdX 53.11dX 44.2HX 36.8 kD> 32.0kD> 26.1kD.This study suggests that there might be a kind of low molecular weight tumor antigens containing conformational determinant expressed on the human ovarian cancer cell membrane. |
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