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

摘要

A murine monoclonal antibody, F10 1 E12, (IgM) wasudproduced against Landschutz ascites tumour cells (LAT)udThis antibody exhibits preferential reactivity on tumourudversus normal cells The antigen against which theudantibody is directed was identified as a cell membraneudglycoprotein of mw 205kD, designated p-205udCharacterization of this antigen by Western immunoblotudanalysis, coupled with enzyme digestion studies, revealedudthat the epitope bound by F10 1 E12 is sensitive to theudeffects of trypsin, pronase and papain. This suggestsudthat an arginine and/or lysine ammoacid residue may beudlocated at this site Lipase and a number ofudglycosidases did not affect the antigen-antibodyudinteraction Neuraminidase was found to destroy thisudinteraction, however, it did lead to the immunodetectionudof a range of other glycoproteins of mw from 40kD toud60kD. This may have been due to the effects of undefinedudproteases in the neuraminidase preparation used.ududThe p-205 of LAT cells was not detectable on a range ofudnormal mouse cells by Western immunoblot analysis,uddespite the fact that certain of these cells, notablyudnormal mouse liver cells, exhibited strong reaction withudF10 1 E12 m solid-phase ELISA studies. This wouldudsuggest that the antigen detected on normal cells isuddifferent to that of LAT cells, or that the antigen asudexpressed by normal cells is more labile than that ofudtumour cells.ududThe glycoprotein p-205 was also detectable by Westernudimmunoblotting analysis using reagent polyclonaludantibodies raised against nigh molecular weightudfucopeptides derived from LAT cell surfaces. Theseudfucopeptides are well characterized m the literature anduddemonstrate a strong association with the neoplasticudstate. This finding may provide some valuable clues asudto the nature of the carbohydrate moieties of p-205.ududIn the course of this project, developments were made mudthe areas of solid-phase ELISA, somatic cell fusionudtechniques using PEG and m the elimination of mycoplasmaudfrom infected hybridomas. These methods and detailedudaccounts of the developments initiated during thisudresearch assignment will also be discussed.