The goal of this project is to develop a flow cytometry detection system (FCDS) for human lymphocytes infected with HIV-1. Optimal conditions for fixation have been established; these not only delineate the presence of intracellular HIV, but also result in inactivation of infectious HIV. Furthermore, primary and secondary antibody reactions have also been characterized. The primary antibody is specific for p24, and the secondary antibody consists of the f(ab')2 fragment of a fluorescein-conjugated, goat anti-mouse immunoglobulin antibody. The system is sensitive enough to detect one HIV-infected cell in 10,000 uninfected cells in experimentally infected human T-lymphoid cell lines. The FCDS is currently being used to examine samples from people known to be at high risk of HIV infection (hemophiliacs and their sexual partners) and from randomly selected blood donors. The system has also been modified to include the use of leu M3 phycoerythrin-labelled monoclonal antibody (CD14) for identifying infected monocytes. Low levels of apparently positive cells have been detected in the leu M3 negative cells. Experiments are currently under way to identify whether or not these cells are infected with HIV.
