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

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Abstract
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. The extent of the conformational change in these isolated complexes was examined by measuring enhancement of the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonic acid and by measuring electrophoretic mobility under native conditions. In all complexes, the fluorescence enhancement was between that observed for native .alpha.2M and that observed for the ternary .alpha.2M-trypsin complex. Likewise, the electrophoretic mobility of the binary complexes was intermediate between that of native .alpha.2M and that of the ternary .alpha.2M-trypsin complex. Thus, formation of a 1:1 .alpha.2M-proteinase complex results in alterations largely confined to one of two functional units within the inhibitor. However, the 1:1 complex only binds between 0.3 and 0.4 mol of additional proteinase. These results suggest that some rearrangement of the binary complex occurs, reducing the ability of the complex to bind additional proteinase molecules. A conformational alteration of the complex once 1 mol of proteinase is bound might be a key factor in determining the binding stoichiometry of .alpha.2M for various proteinases.