Inadequacy of traditional ELISA for screening hybridoma supernatants for murine monoclonal antibodies.
Hybridomas producing anti-creatine kinase (CK) and anti-lactate dehydrogenase (LDH) antibodies were screened by enzyme linked immunosorbant assay (ELISA) with antigen coated onto plastic wells. Out of seven antibodies positive for each isoenzyme of CK, four antibodies failed to bind to the radiolabeled antigen in solution phase radioimmunoassay (RIA) or native antigen in competitive ELISA. Moreover, out of nine antibodies shown reactive with LDH-1 and seven antibodies binding to LDH-5 by ELISA, not a single antibody bound to either radiolabeled or native antigen in solution. Our results along with other recent studies strongly suggest that antigen conformation on solid phase may frequently be different from in solution and that screening by ELISA in which antigen is attached to solid phase is often inappropriate for determining the presence of useful monoclonal antibodies.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D000936:Antigen-Antibody Complex; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000032:analysis / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000032:analysis / Q000276:immunology*; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase; D003402:Creatine Kinase