Production, characterization, and applications of monoclonal antibodies reactive with soybean nodule xanthine dehydrogenase

Abstract

Seven monoclonal antibodies were produced against soybean nodule xanthine dehydrogenase, an enzyme involved in ureide synthesis. Specificity of the seven monoclonal antibodies for xanthine dehydrogenase was demonstrated by immunopurifying the enzyme to homogeneity from a crude nodule extract using antibodies immobilized to Sepharose 4B beads. Each monoclonal antibody was covalently bound to Sepharose 4B beads for the preparation of immunoaffinity columns for each antibody. All seven antibodies were found to be of the IgG1,K subclass. A competitive, indirect enzyme-linked immunosorbent assay demonstrated that two of the seven antibodies shared a common epitope while the remaining five antibodies defined unique determinants on the protein. Rapid, large scale purification of active xanthine dehydrogenase to homogeneity was performed by immunoaffinity chromatography. The presence of xanthine dehydrogenase activity and protein in every organ of the soybean plant was determined. Crude extracts of nodules, roots, stems, and leaves cross-reacted with all seven monoclonal antibodies in an indirect enzyme-linked immunosorbent assay. A positive correlation was observed between the degree of cross-reactivity of a given organ and the level of enzyme activity in that organ. These data demonstrate that xanthine dehydrogenase is not nodule specific. Antigenic variability of xanthine dehydrogenase present in crude extracts from nodules of soybean, wild soybean, cowpea, lima bean, pea, and lupin were detected in the indirect enzyme-linked immunosorbent assay which corresponded to six binding patterns for xanthine dehydrogenase from these plant species. These results correspond well with the epitope determination data which showed that the seven antibodies bind to six different binding determinants on the enzyme.