Monoclonal antibodies to rat Na+,K+-ATPase block enzymatic activity.

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Abstract
A panel of 9 mouse monoclonal antibodies was prepared against purified preparations of rat kidney Na+,K+-ATPase. Selection for specific antibody was based upon the ability of crude hybridoma fluids to inhibit Na+-ATPase activity (using luciferase-linked ATPase assays) and upon antibody binding to both the purified kidney membrane enzyme and to glutaraldehyde-fixed hepatocytes by using standard enzyme-linked immunoadsorbent assays. After immunoaffinity purification, 2 of the antibodies (both of the IgG1 subclass) fully inhibit kidney and liver membrane Na+,K+-ATPase activity with Ki (apparent) values of 30 nM (9-A5) and 600 nM (9-B1). Immunoblots demonstrate directly that 3 different 125I-labeled antibodies (6-4, 9-A5, and 9-B1) bind predominantly to a 94,000 MW protein that comigrates in NaDodSO4/polyacrylamide gels with the fluorescein isothiocyanate-labeled .alpha. subunit of the Na+,K+-ATPase. Indirect immunofluorescence studies with these antibodies on paraformaldehyde-fixed liver slices reveal staining patterns congruent with bile canalicular membrane domains. The antibodies probably exert inhibitory effects by recognizing .alpha. subunits of both liver and kidney Na+ pumps in their native conformations.