Quantification of digoxin by enzyme immunoassay: synthesis of a maleimide derivative of digoxigenin succinate for enzyme coupling.

We synthesized the m-maleimidobenzoyl derivative of digoxigenin-3-0-succinate (through a p-phenylenediamine bridge) as a hapten derivative directed towards coupling to sulfhydryl grouos of beta-galactosidase. Prepared enzyme conjugate had about 97% of the enzyme labeled with the hapten derivative while retaining full enzyme activity. The enzyme immunoassay for digoxin we prepared showed a maximum sensitivity of 30 pg per assay (c.v. = 3%) with minimal cross-reaction with digotoxin (3.8%). Our method for hapten conjugation to beta-galactosidase is highly efficient and is simple and easily replicated.