Digoxin Immunoassay with Monoclonal and Polyclonal Antibodies Using Time-Resolved Fluorometry

Abstract

We describe a nonisotopic heterogeneous competitive immunoassay of digoxin in serum using either Fab fragments of a polyclonal antibody or a high-affinity monoclonal antibody. In the assay, digoxin competes with immobilized digoxin (digoxin:thyroglobulin conjugate) for binding to a biotinylated immunoreactant (Fab or monoclonal). The amount of biotinylated moiety bound to the solid phase (white polystyrene microtiter wells), which is inversely related to the amount of digoxin in the sample, is then quantified by adding streptavidin labeled with the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) in the presence of excess Eu3+. The fluorescent immunocomplex formed is measured directly on the dry solid phase by time-resolved fluorometry. The assay is simple to perform and its characteristics are similar to those of other currently used immunoassay techniques. The Fab fragments and the monoclonal antibody procedure performed equally well on the system. Our results suggest that monoclonal antibodies with high affinity for digoxin can be used for the routine determination of the drug in serum.