Monitoring of intracellular levels of 5′-monophosphate-AZT using an enzyme immunoassay

Abstract We have developed a competitive enzyme immunoassay suitable for routine monitoring of intracellular levels of 5′-monophosphate-AZT (AZT-MP). This assay is performed in 96-well microtiter plates coated with anti-rabbit immunoglobulin antibodies and is based on the use of rabbit polyclonal antibodies raised against an AZT-MP analog and of an AZT-MP/acetylcholinesterase conjugate as tracer. It is very sensitive, with a detection limit close to 0.1 ng/ml (0.2 pmol/ml), and precise (CV 100 pmol/10 6 cells) while, as expected, much lower concentrations were measured in resting PBMCs or MDMs (0.1 to 2 pmol/10 6 cells). The assay constitutes a very convenient tool permitting easy, precise studies of the first step of the intracellular metabolism of AZT leading to the formation of AZT-TP in cultured cells.