Radioimmunoassay for quantitation of 2',3'-didehydro-3'-deoxythymidine (D4T) in human plasma.

2',3'-Didehydro-3'-deoxythymidine (D4T, or stavudine) has been recently approved for the treatment of AIDS. In the present study, a specific and sensitive radioimmunoassay (RIA) was developed for the quantitation of D4T in human plasma. The RIA is a double-antibody competitive binding assay which uses anti-D4T antiserum raised in rabbits as the primary antibody, a tritiated derivative of D4T as the radioactive tracer, and goat anti-rabbit immunoglobulin G as the secondary antibody. No cross-reaction between D4T and various nucleoside analogs was detected. The limit of quantitation of the method approximated 20 ng/ml. Replicate analyses of quality control samples (40 to 3,500 ng/ml) had satisfactory intra- and interassay precision (coefficient of variation, 1.7 to 16.7%) and accuracy (deviation, -9.5 to +21.0). This newly developed RIA was successfully used in the monitoring of plasma drug levels in healthy volunteers receiving an oral dose of D4T.

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

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.