CHARACTERIZATION OF A NEW MONOCLONAL ANTIBODY-F4 DETECTING CELL-SURFACE EPITOPE AND P-GLYCOPROTEIN IN DRUG-RESISTANT HUMAN TUMOR-CELL LINES

Using viable adriamycin resistant human ovarian carcinoma cells 2780AD and colchicine resistant human oral epidermoid carcinoma cells KB-24 as the immunogen in primary and subsequent i.p. immunizations, followe d by i.v. boostings with crude plasma membranes of 2780AD, KB-24, Chin ese hamster lung cells resistant to vincristine DC-3F/VCRd-5L, and res istant to daunorubicin DC-3F/DMXX, we have generated a new murine mono clonal antibody (McAb), designated F4, of IgG1 isotype. McAb F4 reacte d strongly with a cell surface epitope of drug resistant cells and ins ignificantly with their drug sensitive counterparts. Cell surface loca lization of F4 epitope was determined by immunofluorescence and laser scanning confocal imaging system. Results obtained from immunoprecipit ation and immunoblot analyses using F4 and mdr1 P-glycoprotein specifi c McAb JSB-1 demonstrated the reactivity of P-glycoprotein with F4. Th ese results along with those obtained from competitive binding-inhibit ion, chemical modification, and enzyme hydrolysis, revealed that McAb F4 detects an extracellular epitope of P-glycoprotein, and is differen t from other major McAbs directed against P-glycoprotein, e.g. C219, M RK16, JSB-1, HYB-241 and C494. Deduced from the putative structure of mdr1 protein and its orientation in cell membrane, it is proposed that F4 epitope is localized in.or near the 3rd, and/or 6th extracellular transmembrane loops of P-glycoprotein.