SYNTHESIS OF ELECTROACTIVE PROTEIN HYBRID, FEC-BSA-DIG, AND ITS APPLICATION TO A NOVEL HOMOGENEOUS ELECTROCHEMICAL IMMUNOASSAY

A novel homogeneous electrochemical immunoassay using ferrocene- (Fec- ), digoxin- (Dig-) doubleconjugated protein, has been developed. The s ystem is based on the principle that electrochemical activity of the c onjugate is suppressed in the presence of an antibody, and restored in the presence of free digoxin. Hence, a new homogeneous immunoassay ca n be carried out when the electroactivity of the conjugate is determin ed. Here, we synthesize the conjugate by controlling the numbers of fe rrocene and digoxin on individual carrier proteins simultaneously Bovi ne serum albumin (BSA) is selected as a carrier protein to which the e lectrochemical labels and the several hapten molecule are bound by Sch iff's base formation. Since the conjugate synthesized here behaves as a multivalent antigen, and is capable of binding to antidigoxin antibo dy, therefore the antigen having electrochemical activity loses its ac tivity depending on antibody concentration. When the conjugate, in whi ch 39 molecules of ferrocene and 8 molecules of digoxin are bound, is used for the inhibition experiment of electrochemical reaction before and after the immunoreaction, peak height currents, responsible for th e redox reactions of the bound ferrocene, decreased depending on the a dded antibody in a dose-dependent fashion. This system is feasible as a homogeneous electrochemical immunoassay, since the method may provid e a simple and a rapid approach toward a homogeneous assay of bioactiv e substances.