Screening monoclonal antibodies against cell-surface antigens. High frequency of natural antibodies.
Two commonly used methods for screening hybridoma supernatants against cell-surface antigens were performed simultaneously on the supernatant culture fluids of different hybridizations, and compared with the detection of Ig secretion. Supernatants reacting with glutaraldehyde-fixed cells (ELISA (Enzyme-linked immunosorbent assay) on fixed cells) are mostly non-specific for the immunogen; in addition, with this method, only half of the antibodies detected by immunofluorescence are identified. These results can be explained by the frequent occurrence of hybridomas secreting antibodies displaying 'natural antibody' properties, which are strongly reactive with intracellular antigens, and apparently made accessible by the fixation procedure. Since artefacts impair the interpretation of results with ELISA on fixed cells, and complement-mediated cytotoxicity usually as a low yield, membrane immunofluorescence remains the best method for screening hybridoma antibodies.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000276:immunology*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D006825:Hybridomas; D007113:Immunity, Innate; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface