Ouabain-specific antibodies: immunochemical properties and reversal of Na+, K+-activated adenosine triphosphatase inhibition

Abstract

Antibodies with high affinity and specificity for the cardiac glycoside ouabain were raised in rabbits. The antigen used was a conjugate of ouabain linked through its rhamnose moiety to terminal α-amino groups of poly D,L alanyl-human serum albumin. Ouabain-specific antibodies were present as early as 3 wk, and rose steadily in titer over the initial 20-33 wk of immunization. Levels as high as 6.5 mg specific immunoglobulin per ml antiserum were reached in one rabbit at the end of 45 wk. The average intrinsic association constants for ouabain were 1.3 × 10⁹ M^-1 and 1.6 × 10⁹ M^-1 in antisera studied in detail, and there was evidence of restricted heterogeneity of binding site affinities. A high degree of specificity was demonstrated. Significant cross-reactivity occurred only with other cardioactive steroid compounds such as acetyl strophanthidin, digoxin, and digitoxin, while endogenous steroids did not cross-react even when present in 1000-fold excess. A rapid and convenient radioimmunoassay procedure for plasma or urine ouabain concentrations was developed using these antibodies. Competition between ouabain-³H tracer and unlabeled ouabain for specific antibody binding sites allowed the measurement of ouabain concentrations as low as 0.1 ng/ml or less without need for extraction procedures. The high association constants observed in these studies permit antibody reversal of established myocardial effects of ouabain. Both blockade and reversal of ouabain inhibition of canine myocardial microsomal Na⁺, K⁺-activated ATPase by antibody were documented, suggesting a possible mechanism for reversal of cellular effects.