Monoclonal antibody production and the application of monoclonal antibodies to the study of tumour cell membrane antigens

A murine monoclonal antibody, F10 1 E12, (IgM) was produced against Landschutz ascites tumour cells (LAT) This antibody exhibits preferential reactivity on tumour versus normal cells The antigen against which the antibody is directed was identified as a cell membrane glycoprotein of mw 205kD, designated p-205 Characterization of this antigen by Western immunoblot analysis, coupled with enzyme digestion studies, revealed that the epitope bound by F10 1 E12 is sensitive to the effects of trypsin, pronase and papain. This suggests that an arginine and/or lysine ammoacid residue may be located at this site Lipase and a number of glycosidases did not affect the antigen-antibody interaction Neuraminidase was found to destroy this interaction, however, it did lead to the immunodetection of a range of other glycoproteins of mw from 40kD to 60kD. This may have been due to the effects of undefined proteases in the neuraminidase preparation used. The p-205 of LAT cells was not detectable on a range of normal mouse cells by Western immunoblot analysis, despite the fact that certain of these cells, notably normal mouse liver cells, exhibited strong reaction with F10 1 E12 m solid-phase ELISA studies. This would suggest that the antigen detected on normal cells is different to that of LAT cells, or that the antigen as expressed by normal cells is more labile than that of tumour cells. The glycoprotein p-205 was also detectable by Western immunoblotting analysis using reagent polyclonal antibodies raised against nigh molecular weight fucopeptides derived from LAT cell surfaces. These fucopeptides are well characterized m the literature and demonstrate a strong association with the neoplastic state. This finding may provide some valuable clues as to the nature of the carbohydrate moieties of p-205. In the course of this project, developments were made m the areas of solid-phase ELISA, somatic cell fusion techniques using PEG and m the elimination of mycoplasma from infected hybridomas. These methods and detailed accounts of the developments initiated during this research assignment will also be discussed.