Monitoring of intracellular levels of 5'-monophosphate-AZT using an enzyme immunoassay.
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 < 20% from 20 to 0.3 ng/ml). Very low cross-reactivities were observed with AZT and the corresponding di- and triphosphate derivatives as well as with other related nucleotides and nucleosides. The validity of the assay was demonstrated by measuring intracellular concentrations of AZT-MP in peripheral blood mononuclear cells (PBMCs) and in monocyte-derived macrophages (MDMs) cultured in the presence of various concentrations of AZT (from 0.01 microM to 10 microM). We observed very high levels of AZT-MP in stimulated (PHA + IL2) PBMCs (> 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.
Goujon
L
L
CEA, Département de Recherche Médicale, CEA Saclay, Gif sur Yvette, France.
Brossette
T
T
Dereudre-Bosquet
N
N
Creminon
C
C
Clayette
P
P
Dormont
D
D
Mioskowski
C
C
Lebeau
L
L
Grassi
J
J
D054306:Dideoxynucleotides; D018023:HIV Seronegativity; D006241:Haptens / Q000032:analysis / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D012680:Sensitivity and Specificity; D013942:Thymine Nucleotides / Q000032:analysis* / Q000276:immunology; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology
D054306:Dideoxynucleotides; D006241:Haptens; D013942:Thymine Nucleotides; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine