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Anti-digoxin monoclonal antibodies are a useful model for basic immunochemical studies, for investigation of endogenous digoxin-like substances and in immunoassay for cardiac glycosides. The complete phenotypic characterization is a requisite for the selection of antibodies with desired binding parameters for different purposes. Twenty-two high-affinity monoclonal antibodies specific for digoxin were obtained in two fusion experiments. Treatment of antigen-antibody complex with potassium thiocyanate (KSCN) and absorption ELISA were used for the selection of high-affinity antibodies at the earliest stages of hybridoma growth. The true affinity constants of selected antibodies were determined in ELISA. They had proven to vary between 10(-7) and 10(-10) M. The fine specificity of 21 anti-digoxin monoclonal antibodies was determined by cross-reactivity experiments with 25 structurally related compounds. Cardiac glycosides, digoxin metabolites, endogenous steroids and spironolactone were used in the elucidation of the antigenic recognition pattern of antibodies. The elucidation of the binding characteristics of anti-digoxin monoclonal antibodies makes possible the selection of antibodies possessing binding characteristics appropriate for a wide range of designations.

The specificity of various monoclonal antidigitoxin antibodies was characterized using 6 cardiac glycoside analogs. Spleen cells from BALB/c mice, immunized with BSA- or KLH-digitoxin conjugates, were fused with NS1 myeloma cells, and antibody-producing hybrids were identified by radioimmunoassay. Twenty-one monoclonal antidigitoxin-specific antibodies were obtained, 10 of which were cloned and characterized for affinity and specificity. All the antibodies had a high affinity constant, ranging from 8.10(8) to 2.5.10(10) 1/M. On the basis of their binding specificities, the antibodies could be classified into 3 groups: the first contained 7 antibodies exhibiting high cross reactivity (42-100%) with digitoxigenin, whereas the second and third groups did not recognize this analog (cross-reactivity of 1%). In the former group, the absence of the sugar moiety only slightly affected the binding reaction, although for the two other groups, this structure did appear to be involved in antibody recognition. Changes in the functional groups of the hapten molecule led to considerable changes in the antibody-antigen reaction. For all the antibodies except one, saturation of the lactone ring considerably affected binding. These results demonstrated that monoclonal antibodies of different specificities with respect to both the steroid backbone and the sugar moiety of digitoxin can be induced using a digitoxin-protein conjugate.

We compared the primary response in vitro with the secondary response in vivo of A/J inbred and CD-1 outbred mice to digoxin-HSA. The frequencies of hybrid formation and growth, and of the hybridomas that secreted antibody to digoxin were similar in both strains regardless of the immunization procedure. The patterns of cross-reactivity of the monoclonal antibodies to two compounds structurally related to digoxin (ouabain and digitoxin) were likewise similar for both strains and immunization procedures.