Comparison of non-digitalis binding properties of digoxin-specific Fabs using direct binding methods.
Digibind and DigiFab are commercial formulations of polyclonal, ovine, digoxin-specific Fabs in clinical use for treatment of digoxin intoxication. Of interest for extending its use to other clinical indications, Digibind has also been reported to neutralize the effect of endogenous digoxin-like molecules, including ouabain, that are linked to clinical disorders ranging from preeclampsia to congestive heart failure. Although Digibind and DigiFab are equivalent in their digoxin-binding activity, the antigens used to produce these Fabs are different. We therefore explored, using native (3)H-digoxin and (3)H-ouabain in four different types of solution-phase binding methods, whether they might exhibit different profiles with respect to ouabain and other digoxin-like factors. Consistent with previous results, both Fab preparations bound digoxin with the same affinities and capacities. However, (3)H-ouabain was found to bind with high affinity only to Fab sub-populations present in both products. Interestingly, this sub-population was twice as large for Digibind compared to DigiFab. Competition experiments also showed differences in specificity within Fab sub-populations. Therefore, the equivalence in digoxin-binding activity of the two Fab preparations does not extend to ouabain-binding capacity and Fab specificity, with implications for clinical differentiation between the preparations in treatment of disorders related to control of non-digoxin cardenolides. The existence of a small but perhaps clinically relevant sub-population of antibodies was detected using specific radioligands. This sub-population could not have been detected nor quantified using standard cross-reactivity in an ELISA assay.
Department of Biochemistry, Cardiovascular and Urogenital Center of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USA. Mark.A.Pullen@GSK.com
Mark A
MA
Pullen
Mark R
MR
Harpel
Theodore M
TM
Danoff
David P
DP
Brooks
D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D001667:Binding, Competitive; D002018:Bufanolides / Q000276:immunology / Q000378:metabolism; D004077:Digoxin / Q000276:immunology* / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000276:immunology* / Q000378:metabolism; D010042:Ouabain / Q000276:immunology* / Q000378:metabolism
D002018:Bufanolides; D007140:Immunoglobulin Fab Fragments; C050199:digoxin antibodies Fab fragments; C093896:marinobufagenin; D010042:Ouabain; D004077:Digoxin