Rat Monoclonal Antibody Preparation, Identification, Gene Cloning And Antigenic Analysis And Anti-tumor Effect Of The Drug Conjugates

It has been noted that antibodies from rats possess characters distinct from those of the mouse. Ninety five percent light chain of rat antibody are k type. Therefore, it is possible to use mouse against rat Igk-1a antibody to separate and purify antibody secreted by Igk-1a allele-bearing hybridoma from the immunoglobulins of inoculated Igk-1b rats. However, it is well-known that the preparation of rat hybridomas is of considerable difficulties. In the present study, human immunoglobulin IgG, IgM and horseradish peroxidase were employed to immunize LOU/C rats. The spleen cells were then fused with rat myeloma IR983F cells. A number of monoclonal antibodies against human IgG, IgM, or horseradish peroxidase were obtained, and the fusion rates were about 70%.Selected hybridomas, under proper cultural conditions, exhibited long-term growth pattern and steadily produced antibodies against designated antigens. Ascites from hybridoma-inoculated animals contained highly active and concentrated antibodies. The isolation methods were simple and efficient. A number of the clones displayed high affinity to their antigens by ELISA assay with the avidity between 10⁷-10¹⁰ M^-1. Our results suggest that the rat hybridoma technique could be developed to obtain a large quantity of high affinity monoclonal antibodies.By using the methods described above, BEL-7402 cells (human liver cancer cells) were served as target antigen to immunize LOU/C rats, followed by fusion of spleen cells and rat IR983F myeloma cells. The resulting clone (3A5) secreted steadily anti-human liver cancer McAb. Immuno-reactivity by ELISA revealed specific positive responses to human liver cancer BEL-7402, 7721 and Q3 cells but no reaction to human epidermoid cancer KB cells, human fetal lung 2BS cells and normal mouse liver cells. Immunohistochemical...