Quantitation of 3'-amino-3'-deoxythymidine (AMT), a toxic catabolite of 3'-azido-3'-deoxythymidine (AZT), by competitive ELISA.

Abstract

In the present study, a competitive ELISA technique was developed to specifically quantitate 3'-amino-3'-deoxythymidine (AMT), a toxic catabolite of 3'-azido-3'-deoxythymidine (AZT) detected in serum from AZT-treated patients. In order to eliminate cross-reacting AZT, serum sample was extracted with ethylacetate and then AMT was acetylated (Ac-AMT). A 5'-hemisuccinate-AMT-horseradish peroxidase conjugate was used as a tracer in the presence of anti-AMT rabbit antibodies which were raised against a 5' hemisuccinate-AMT-bovine serum albumin immunogen. Bound/free separation was achieved with an anti-rabbit IgG mouse monoclonal antibody insolubilized onto a microtiter plate. The limit of quantification of Ac-AMT was as low as 0.4 ng/ml in serum samples. This ELISA technique was applied for monitoring AMT plasma levels in patients receiving AZT therapy. The intra and inter-individual variations of the AZT/AMT plasma concentration ratios underlined the need for such a specific test in studying the formation of this toxic catabolite.