Functional heterogeneity of monoclonal antibodies obtained using different screening assays.
Two alternate screening methods have enabled the detection of monoclonal antibodies with different specificities toward the lysosomal enzyme alpha-mannosidase of Dictyostelium discoideum. Spleen/myeloma hybrid cell cultures were screened for antibody production by separate assays: an indirect enzyme-linked immunoadsorbent assay (ELISA) based on the antibody binding to enzyme adsorbed on plastic, and a direct assay of the antibodies' ability to precipitate enzyme activity with fixed Staphylococcus aureus cells (Pansorbin). Fourteen stable antibody-producing cell lines resulted from a single fusion; these fell into three distinct classes based on their screening characteristics. A group of eight were positive in both assays, and these immunoprecipitated a 140,000 Mr precursor form of alpha-mannosidase in addition to the 58,000 and 60,000 Mr mature enzyme subunits from [35S]methionine-labeled total secreted protein preparations. Two of the antibodies were positive only in the immunoprecipitation assay; these failed to precipitate the 140,000 Mr precursor. The third class consisted of four antibodies that were positive only in the ELISA method. These exclusively recognized an altered conformation of the enzyme (precursor and mature forms) that was immobilized either on plastic or on nitrocellulose paper. In addition, only members of this class were able to bind to immobilized fragments of protease-treated enzyme. The implications of these findings for the general design of monoclonal antibody screenings and for the alternative structures of this enzyme are discussed.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000302:isolation & purification*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D055598:Chemical Phenomena; D011232:Chemical Precipitation; D002621:Chemistry; D004023:Dictyostelium; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007120:Immunochemistry; D008361:Mannosidases / Q000276:immunology*; D011487:Protein Conformation; D043323:alpha-Mannosidase
D000911:Antibodies, Monoclonal; D008361:Mannosidases; D043323:alpha-Mannosidase