A solid-phase radioimmunoassay using digitonin-permeabilized cells to screen surface or intracellular membrane-bound antigens.
Membrane-bound receptor or enzyme distribution between cell surface and cell interior can be determined using the non-ionic detergent digitonin. A solid-phase radioimmunoassay using myocardial cells from newborn rats was performed to screen hybridoma culture supernatants and the cells were rendered permeable with increasing concentrations of digitonin (0-0.1%). This was achieved in 5 min at 37 degrees C and did not require the continuous presence of detergent. A characteristic amount of cytoplasmic protein (approximately equal to 45%) was released with subunit molecular weights of up to 200,000. This allowed exogenous molecules such as immunoglobulin G to gain access to the intracellular targets. The binding to rat myocardial cells of 36 monoclonal antibodies was examined by this procedure. The binding assays were carried out for 30 min at 37 degrees C using cells previously treated for 10 min with 0.05% of digitonin. This appears to be a simple and rapid method of screening and selecting the hybridoma culture supernatants.
D000818:Animals; D000831:Animals, Newborn; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000032:analysis*; D000954:Antigens, Surface / Q000032:analysis*; D002462:Cell Membrane / Q000032:analysis; D002463:Cell Membrane Permeability; D004072:Digitonin; D004285:Dogs; D006352:Heart Ventricles / Q000032:analysis; D007425:Intracellular Membranes / Q000032:analysis*; D051379:Mice; D008815:Mice, Inbred Strains; D009206:Myocardium / Q000032:analysis; D011863:Radioimmunoassay / Q000379:methods; D012508:Sarcolemma / Q000032:analysis
D000911:Antibodies, Monoclonal; D000941:Antigens; D000954:Antigens, Surface; D004072:Digitonin