MONITORING OF INTRACELLULAR LEVELS OF 5'-MONOPHOSPHATE-AZT USING AN ENZYME-IMMUNOASSAY

We have developed a competitive enzyme immunoassay suitable for routin e 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 po lyclonal antibodies raised against an AZT-MP analog and of an AZT-MP/a cetylcholinesterase conjugate as tracer. It is very sensitive, with a detection Limit close to 0.1 ng/ml (0.2 pmol/ml), and precise (CV < 20 % from 20 to 0.3 ng/ml). Very low cross-reactivities were observed wit h AZT and the corresponding di- and triphosphate derivatives as well a s with other related nucleotides and nucleosides. The validity of the assay was demonstrated by measuring intracellular concentrations of AZ T-MP in peripheral blood mononuclear cells (PBMCs) and in monocyte-der ived macrophages (MDMs) cultured in the presence of various concentrat ions of AZT (from 0.01 mu M to 10 mu M). We observed very high levels of AZT-MP in stimulated (PHA + IL2) PBMCs (> 100 pmol/10(6) cells) whi le, as expected, much lower concentrations were measured in resting PB MCs 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. (C) 1998 Elsevier Science B.V. All rights reserved .