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Antimicrobial Agents and Chemotherapy, January 2004, p. 104-109, Vol. 48, No. 1
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.1.104-109.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Sensitive Enzyme Immunoassay for Measuring Plasma and Intracellular Nevirapine Levels in Human Immunodeficiency Virus-Infected Patients

Stéphane Azoulay,1 Marie-Claire Nevers,2 Christophe Créminon,2 Laurence Heripret,3 Jacques Durant,3 Pierre Dellamonica,3 Jacques Grassi,2 Roger Guedj,1 and Danièle Duval1*

Laboratoire de Chimie Bio-Organique, Université de Nice-Sophia Antipolis,1 Service des Maladies Infectieuses et Tropicales, Hôpital Archet, Nice,3 CEA, Service de Pharmacologie et d'Immunologie, DRM, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France2

Received 12 May 2003/ Returned for modification 18 September 2003/ Accepted 7 October 2003

We have developed an enzyme immunoassay to measure nevirapine (NVP) in plasma and peripheral blood mononuclear cells. Anti-NVP polyclonal antibodies were raised in rabbits by using a synthetic NVP derivative coupled to keyhole limpet hemocyanin as the immunogen, and the enzyme tracer was prepared by chemically coupling the NVP derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates with a 100-pg ml-1 limit of detection and thus ~100 times more sensitive than previously published techniques. The plasma assay was performed directly without extraction (in this case, a 500-pg ml-1 limit of detection was observed) on a minimum of 30 µl of plasma. This assay shows good precision and efficiency, since recovery from human plasma and cell extracts spiked with NVP ranged between 87 and 104%, with coefficients of variation of <10%. A pharmacokinetic analysis of plasma NVP was performed for seven patients infected with human immunodeficiency virus (HIV), and it gave results similar to published findings. Intracellular concentrations of NVP were measured in cultured human T-lymphoblastoid cells and peripheral blood mononuclear cells from HIV-infected patients. The results indicated a very low intracellular/extracellular concentration ratio (0.134), thus demonstrating the absence of intracellular drug accumulation. This is the first intracellular assay of a nonnucleoside reverse-transcriptase inhibitor, and this method could be useful in monitoring plasma and intracellular NVP levels in HIV-infected patients.

* Corresponding author. Mailing address: Laboratoire de Chimie Bio-Organique UMR 6001, Faculté des Sciences, Université de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France. Phone: 33 4 92 07 61 15. Fax: 33 4 92 07 61 51. E-mail: duvald{at}unice.fr.

Antimicrobial Agents and Chemotherapy, January 2004, p. 104-109, Vol. 48, No. 1
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.1.104-109.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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