Identification of a monoclonal antibody specific for a neoantigenic determinant on alpha 2-macroglobulin: use for the purification and characterization of binary proteinase-inhibitor complexes.
A monoclonal antibody was obtained from the fusion of spleen cells of mice, immunized with methylamine-treated alpha 2-macroglobulin (alpha 2M), with the myeloma cell line P3-X63-Ag8.653. A competitive binding assay demonstrated that the antibody was specific for a neoantigen expressed on alpha 2M when the inhibitor reacts with proteinases or with methylamine. When immobilized, the monoclonal antibody retained its ability to specifically bind alpha 2M-proteinase complexes or methylamine-treated alpha 2M, both of which could be quantitatively recovered from the immunoaffinity column by lowering the pH to 5.0. Binary alpha 2M-proteinase complexes of trypsin, plasmin, and thrombin, prepared by incubating large amounts of alpha 2M with a small amount of enzyme, were isolated by immunoaffinity chromatography. Each purified complex was characterized with regard to proteinase content, extent of alpha 2M subunit cleavage, extent of thiol ester hydrolysis, and extent of conformational change. Each complex contained 0.8-0.9 mol of proteinase/mol of inhibitor. In the alpha 2M-thrombin, alpha 2M-plasmin, and alpha 2M-trypsin complexes, approximately 50%, 60%, and 75% of the subunits are cleaved, respectively. Titration of sulfhydryl groups revealed that all purified binary complexes contained 2 +/- 0.5 mol of thiol/mol of complex, suggesting that each complex retains two intact thiol ester bonds. When the purified complexes were incubated with excess trypsin or with methylamine, an additional 1-2 mol of sulfhydryl/mol of complex could be titrated.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001665:Binding Sites; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D000939:Epitopes / Q000032:analysis*; D005341:Fibrinolysin / Q000378:metabolism; D007074:Immunoglobulin G / Q000302:isolation & purification; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000276:immunology* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D000511:alpha-Macroglobulins; D004228:Dithionitrobenzoic Acid; D010447:Peptide Hydrolases; D014357:Trypsin; D013917:Thrombin; D005341:Fibrinolysin