Monoclonal antibodies that distinguish between two related digitalis glycosides, ouabain and digoxin

The exogenous digitalis glycosides, ouabain and digoxin, have been widely u sed in humans to treat congestive heart failure and cardiac arrhythmias, Se veral reports have also pointed to the existence of endogenous ouabain- and digoxin-like compounds, hut their precise roles in mammalian physiology an d various disorders of the circulation are not clear. In an attempt to prod uce specific Abs for the purification and identification of endogenous ouab ain-like compounds, somatic cell fusion was used to produce mAbs specific f or ouabain, Our attempts to produce ouabain-specific mAbs were unsuccessful when ouabain was coupled to exogenous proteins such as bovine gamma-globul ins, BSA, and human serum albumin. However, when ouabain was coupled to an Ab of A/J mice origin and the same strain of mouse was used for immunizatio n with ouabain-ab conjugate, three Abs (1-10, 5A12, and 7-1) specific for o uabain were obtained. In assays of fluorescence quenching and saturation eq uilibrium with tritiated ouabain, Ab 1-10 exhibited 200 nM affinity for oua bain, These three mAbs are distinguished from existing Abs to ouabain and d igoxin by their specificity for ouabain and lack of cross-reactivity with d igoxin. Specificity studies showed that the loss of cross-reactivity was co rrelated with the presence of a hydroxyl group at either position 12 beta ( digoxin) or 16 beta (gitoxin) of the steroid ring. These Abs can be used to develop assays for detection and characterization of ouabain-like molecule s in vivo.