Development of a monoclonal antibody to PIVKA-IX, PIVKA-X and study on the mechanism of production of PIVKA by hepatocellular carcinoma

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l. Development of monoclonal antibodies to PIVKA-IX and PIVKA-XThe currently available monoclonal antibody to PIVKA-II shows a difference in reactivity according to the degree of carboxylation of the PIVKA-II antigen. In order to perform a detailed study on PIVKA-II, a monoclonal antibody which reacts with all of the PIVKA-II's is required. Accordingly, we changed the initial plan of developing monoclonal antibodies to PIVKA-IX and X to developing monoclonal antibodies to all of the PIVKA-II's and PIVKA-IX, To purify native PIVKA-II and PIVKA-IX we stored the culture supernatant of cultured hepatoma cell huH-2. The method of developing monoclonal antibodies to F-II and F-IX was to obtain antibody-producing clones by fusing spleen cells of intraperitoneally immunized mice and myeloma cells by polyethylene glycol. To check the clones ELISA was performed using assay plates coated with F-II and F-IX. Sixteen antibody-producing clones were obtained with F-II and 24 were obtained with F-IX. These cells were injected intraperitoneally into mice, their ascites were purified and an affinity column againgi F-IX was made. Using this column we plan to develop a monoclonal antibody to PIVKA-IX by purifying the stored culture supernatant. We are also making an affinity column for the development of an antibody to all of the PIVKA-II's.2. Study on the mechanism of production of PIVKAWe measured the activity of gamma -glutamyl carboxylase by solubilizing microsomes obtained from hepatocellular carcinoma (HCC) tissue by ultracentrifugation. There was no defect in this enzyme in HCC and high activities were shown indicating overproduction of prothrombin precursors. When we measured vit. K levels of HCC tissue by HPLC, there was a decrease in uptake of vit. K by HCC tissue when there was an overload of vit. K. We also established a new experimental model by transplanting cultured hepatoma cells into nude mice. This model was useful in studying the sensitivity of HCC to vit. K.