RAPID HYBRIDOMA SCREENING METHOD FOR THE IDENTIFICATION OF MONOCLONAL-ANTIBODIES TO LOW-ABUNDANCE CYTOPLASMIC PROTEINS

Screening assays are the most time-consuming and labor-intensive part of generating monoclonal antibodies (MAbs). Antibodies identified by e nzyme-linked immunosorbent assay (ELISA) screening often are not suita ble for their intended application such as immunofluorescence staining . We describe here a rapid and efficient flow cytometric screening pro cedure for the identification of MAbs directed against low-abundance c ytoplasmic proteins, in our case, the pro-apoptotic molecule Bim. Cell s from all equal mixture of a parental cell line and a subline express ing Bim were fixed, permeabilized and incubated with hybridoma superna tants. The supernatants were derived from a fusion of Sp2/0 plasmacyto ma cells and spleen cells from a rat immunized with recombinant glutat hione-S-transferase nse (GST)-Bim(L) fusion protein. Secondary stainin g with fluorochrome-labeled anti-rat Ig antibodies allowed detection o f clones expressing Bim-specific antibodies. The screening procedure w as rapid and efficient, nod most monoclonal antibodies identified were proven to De useful for immunofluorescence staining and other applica tions.