| Carcinoembryonic Antigen(CEA) is one kind of glycoprotein with high molecular weight (200KD), existing in the blood or on the surface of some tumor cells. Because the level of CEA of fetus and some tumor patients is far higher than that of normal adults, it is considered as a special molecular marker of related tumors. Now anti-CEA monoclonal antibodies have been applied to immunodiagnosis, immunoscintigraphy and immunotherapy of CEA related tumors. Anti-CEA monoclonal antibody, c50 strain, was invented by NVSI in 1984. and had been proved by the clinical tests to be of high affinity, and have no cross-reaction with human leukocytes and myelocytes. These features promised a good application in immunoscintigraphy and immunotherapy of CEA positive tumors in vivo. Preparing for producing c50 antibodies with the method of culturing c50 cells in vitro in the future, we studied the feature of antibody secretion and culturing strategy of c50 cells.We first developed a special ELISA method to detect the concentration of c50 antibody in the supernatant. And with the use of protein G affinity chromatography, we deleted the bovine IgG from the cell culture medium. After culturing c50 cells with this kind of medium (-GFCS medium) in vitro successfully, c50 antibodies existing in the supernatant had been purified by protein G affinity chromatography easily, the products of which were not. contaminated with any bovine IgG. Using the method of chromosome counting, we found that some c50 cells (16%)lost more chromosomes than normal hybridoma cells. Using the method of limiting dilution, we subcloned c50 cells, and found that the positive percentage of c50 cell line was about 84%, much lower than normal (above 95%). These two results suggested that genetic unsteadiness might lead to the low antibody secretion of c50 cells cultured in vitro.The kinetics of cell growth and antibody secretion suggested that the antibody production of c50 cell line was relatively low(no more than 20 n g/ml)and unstable. During one cycle of cell growth, the antibody production increased or deceased with the changing of live cell density, although the special growth velocity of cells did not change markedly. We also found that raising the beginning density of c50 cells, while culturing them in vitro, could not only increase both the maximum density of live cells and the maximum concentration of c50 antibody in supernatant, but also make them emerging earlier. The minimum percentage of bovine serum of the medium to support the normal growth and antibody secretion of c50 cell in vitro was proved to be 5%. Raising the percentage of bovine serum could also make the maximum of antibody secretion emerging earlier. The conclusion of the positive relativity between live cell density and antibody secretion was also confirmed by the result of semi-continuous propagation with the use of 0. 5L spinner-flask.After culturing the cells in vitro for more than fifteen days, the level of c50 antibody in supernatant decreased markedly. When we returned these c50 cells back into the belly of allogenetic mouse and took them out after fifteen days, their ability to produce c50 antibody was recovered to the maximum level (17ug/ml), however, then lost again in more than fifteen days.To confirm the environmental affection to antibody secretion of c50 cell line, we added a kind of CpG ODN into the medium in which those disable c50 cells was cultured. As a result, the ability of antibody secretion was improved markedly, as well as the percentage and the density of live cells. The work prescribed above displayed: the low positive percentage of c50 cell line caused by the abnormal loss of chromosomes during the process of proliferation of the cells, might lead to the low and unstable antibody secretion. Either to keep high density of live cells or regulate the synthesis and secretion of c50 antibody by optimizing the environmental factors was proved to be an effective way to improve the antibody concentration. |
PDF Full Text Request