High-affinity monoclonal antibodies to the cardiac glycoside, digoxin.

Abstract High-affinity anti-digoxin antibodies isolated from antisera are known to reverse the pharmacologic and toxic effects of the widely used cardiac glycoside, digoxin. The technique of somatic cell fusion has the potential of providing an unlimited supply of high-affinity monoclonal antibodies specific for digoxin that are suitable for therapeutic and diagnostic use. We produced seven high-affinity monoclonal anti-digoxin antibodies using this technique. Spleen cells from A/J mice immunized with digoxin coupled to human serum albumin were fused with one of three myeloma cell lines (45.6TG1.7, NS1, or Sp2/0). Antibody production in subcloned cell lines was augmented from micrograms per milliliter in culture media to milligrams per milliliter quantities in ascites. Purification from ascites was accomplished by several means, including affinity chromatography on ouabainamine-Sepharose columns and ion-exchange chromatography on DEAE cellulose. Each hybridoma antibody was analyzed for affinity and specificity by radioimmunoassay (RIA) based on the adsorption of the digoxin hapten to dextran-coated charcoal. All of the monoclonal antibodies demonstrated high affinity constants (2 X 108 to 7 x 109 M–1). Of the seven antibodies analyzed for specificity to structurally related cardiac glycosides, three demonstrated unique sets of inhibition curves. The other four antibodies showed nearly identical patterns in their inhibition profiles. These same four antibodies gave identical patterns on isoelectric focusing. Two hybridomas, Dig 26-10 and Dig 35-20, were shown to be as effective in a clinical RIA for digoxin as sheep anti-digoxin sera and as the anti-digoxin antibody supplied in a commercially available kit. Analysis of the fine specificity for antigen of each of three monoclonal antibodies revealed small differences in the probable orientation of digoxin in the antibody combining site. Characterization of these and other high-affinity monoclonal anti-digoxin antibodies with regard to their variable region amino acid sequences and their antigenic fine specificity for related cardiac glycosides will provide a model system for the study of structure-function relationships between antibody combining site and specific antigenic determinants.