Development of a New Cartridge Radioimmunoassay for ...

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May 19, 1998 - ment by radioimmunoassay using a newly developed anti-3TC serum. ... by RIA. In the present study, we report on the development of.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 1998, p. 2656–2660 0066-4804/98/$04.0010 Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Vol. 42, No. 10

Development of a New Cartridge Radioimmunoassay for Determination of Intracellular Levels of Lamivudine Triphosphate in the Peripheral Blood Mononuclear Cells of Human Immunodeficiency Virus-Infected Patients BRIAN L. ROBBINS,1 THU T. TRAN,1 FRANK H. PINKERTON, JR.,1 FATIMA AKEB,2 ROGER GUEDJ,2 JACQUES GRASSI,3 DANNY LANCASTER,4 1,5 AND ARNOLD FRIDLAND * Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee 381051; Laboratoire de Chimie Bioorganique, UPRES-A 6001 CNRS, Universite´ de Nice-Sophia Antipolis, 06108 Nice, Cedex 22, and Commissariat a ` l’Energie Atomique, Service de Pharmacologie et d’Immunologie, C.E.-Saclay, F-91191 Gif sur Yvette Cedex,3 France; Methodist Hospital, Memphis, Tennessee 381044; and Department of Pharmacology, University of Tennessee, Memphis, Tennessee 381015 Received 2 February 1998/Returned for modification 19 May 1998/Accepted 25 July 1998

A new sensitive method for the measurement of lamivudine triphosphate (3TC-TP), the active intracellular metabolite of lamivudine in human cells in vivo, has been established. The procedure involves rapid separation of 3TC-TP by using Sep-Pak cartridges, dephosphorylation to 3TC by using acid phosphatase, and measurement by radioimmunoassay using a newly developed anti-3TC serum. The radioimmunoassay had errors of less than 21% and a cross-reactivity of less than 0.016% with a wide variety of other nucleoside analogs. The limit of quantitation of the assay for intracellular 3TC-TP was 0.195 ng/ml (0.212 pmol/106 cells), and a cell sample of only 4 million cells was ample for the assay. This procedure, combined with our previously developed method for measuring zidovudine (ZDV) metabolite levels, proved capable of measuring 3TC-TP, ZDV monophosphate (ZDV-MP) and ZDV triphosphate (ZDV-TP) in human immunodeficiency virus (HIV)-infected subjects treated with combination 3TC and ZDV therapy. In seven subjects, intracellular 3TC-TP levels ranged from 2.21 to 7.29 pmol/106 cells, while intracellular ZDV-MP and ZDV-TP levels ranged from