Production of monoclonal antibody against electrophoretically purified RNA polymerase II subunits using in vitro immunization

A procedure has been developed for the production of MAb against weakly immunogenic subunits of a multisubunit enzyme. This procedure takes into account the problems of insufficient antigen, single epitope immunodominance and the difficulty of mapping non-sequential determinants. Small quantities of mammalian RNA polymerase II subunits were purified by SDS-polyacrylamide gel electrophoresis and were used to immunize splenocytes in vitro. After fusion with plasmacytoma cells, the hybrid cells were cloned and screened by ELISA utilizing native RNA polymerase II. This procedure is biased towards the production of MAb directed against sequential epitopes accessible on the native enzyme. Monoclonal antibodies, produced by in vitro immunization, were shown to be useful in protein transblot analyses, to inhibit enzyme activity in vitro and to have binding affinities comparable with MAbs produced by in vivo immunization.