Abstract
Nine hybridoma cell lines secreting antibodies against the maize leaf nitrate reductase have been distinguished by reciprocal competition for binding to the antigenic site. Inhibition of enzymatic activities, and western blots of native enzyme and denatured subunits revealed different behaviors of individual antibodies towards the antigen. Two classes of monoclonal antibodies are inhibitory of NADH and methyl viologen nitrate reductase activities, but only one affects also NADH cytochrome c reductase activity. The associated epitopes are sensitive to antigen conformation. Among the 4 other classes, one is specific for the native conformation of the molecule, another binds more strongly to the denatured antigen, and two recognize equally well the two forms.
Abbreviations: BSA, bovine serum albumin; EDTA, ethylenediaminetetraacetic acid, Na salt; SDS, sodium dodecyl sulfate; ELISA, enzyme linked immunosorbent assay; PBS, phosphate buffered saline, pH 7.2; PBS-T, PBS containing 0.05% Tween 20; PAGE, polyacrylamide gel electrophoresis
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