Dose-dependent reversal of digoxin-inhibited activity of an in vitro Na+K+ATPase model by digoxin-specific antibody.

We investigated the potency of digoxin-specific Fab fragments to reverse digoxin-induced Na+K+ATPase inhibition in rat brain microsomes according to (a) the extent of initial inhibition of Na+K+ATPase and (b) the neutralizing dose of antibody. Mathematical analysis of the digoxin concentration-Na+K+ATPase inhibition curve supports the existence of 2 digoxin sensitive Na+K+ATPase isoforms. The IC50 was 1.3 x 10(-4) M and 2.5 x 10(-8) M for the low (alpha 1) and high (alpha 2) digoxin affinity isoenzyme, respectively. The reversal of digoxin-induced Na+K+ATPase inhibition was dependent on the digoxin-specific Fab concentration. The maximal effect was observed when the Fab:digoxin ratio was stoichiometrical and addition of an excess of antibodies did not result in a complete reversal of inhibition at the 4 digoxin concentrations studied. This simple and rapid in vitro model will be a useful tool to predict the efficacy of a new generation of antibodies.