E-Book Overview
This collection of cutting-edge techniques is essential for studying the molecular biology, virology, and immunology of the HIV virus. The methods cover a broad range of research interests, including quantitation of viral genomes, HIV promoter function, B-cell epitope mapping, viral coreceptor usage, and measurements of T-cell function. Special emphasis is accorded to studying those viral and host immune responses to infection that will be critical to the design of effective preventive vaccines. HIV Protocols is the first HIV methods book to concentrate equally on virology, molecular biology, and immunology, as well as to incorporate methods on chemokine receptor structure and function.
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1 Isolation and Expansion of HIV from Cells and Body Fluids by Coculture James R. Lane 1. Introduction HIV can be recovered from infected patients at all stagesof the disease spectrum. Typically, the quantity of biologically active virus, or viral protein, in body tissues is below the level of direct detection by either antigen capture or reverse transcrtptase assays.Consequently, the virus must be expanded in culture. This may be achieved by the coculttvation of pattent material with mitogen-stimulated peripheral blood mononuclear cells (PBMCs) from normal, healthy donors. These cocultures are then maintained by regularly scheduled mterleukin-2 (IL-2) supplemented medium replacement, and the periodic addition of freshly stimulated normal donor PBMCs. Durmg this cocultivation period, culture fluids are harvested at regular intervals and tested for the presence and subsequent replication of HIV. Cultures fatling to demonstrate evidence of vnus expression within 35 d are usually terminated. It is well recognized that the efficiency of virus isolation is dependent on specimen type. Patient PBMCs represent a useful source of patient vnus, however, isolates can be obtained from suc