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

L. PIVKA-IX和PIVKA-X单克隆抗体的开发目前可用的PIVKA-II单克隆抗体根据PIVKA-II抗原的羧化程度显示出反应性差异。为了对PIVKA-II进行详细研究，需要与所有PIVKA-II反应的单克隆抗体。因此，我们将开发针对PIVKA-IX和X的单克隆抗体的初始计划改变为开发针对所有PIVKA-II和PIVKA-IX的单克隆抗体。为了纯化天然PIVKA-II和PIVKA-IX，我们储存培养的肝癌细胞huH-2的培养上清液。制备抗F-II和F-IX单克隆抗体的方法是将腹腔免疫小鼠的脾细胞与骨髓瘤细胞用聚乙二醇融合获得抗体产生克隆。为了检查克隆，使用用F-II和F-IX包被的测定板进行ELISA。用F-II获得16个抗体产生克隆，用F-IX获得24个。将这些细胞腹腔注射到小鼠体内，纯化其腹水，并制成抗F-IX的亲和柱。使用该柱，我们计划通过纯化储存的培养物上清液来开发针对PIVKA-IX的单克隆抗体。我们还在制作一种亲和柱，用于开发针对所有PIVKA-II的抗体。2. PIVKA产生机制的研究我们用超离心法从肝细胞癌（HCC）组织中获得微粒体，通过溶解微粒体来测定γ-谷氨酰羧化酶的活性。在HCC中这种酶没有缺陷，并且显示出高活性，表明凝血酶原前体过度产生。当我们测量维生素时。肝癌组织中维生素K水平降低，维生素摄取减少。K通过HCC组织时，有一个过载的维生素。K.我们还建立了一种新的肝癌细胞裸鼠移植瘤模型。该模型可用于研究肝癌对维生素的敏感性。K.

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.