DETECTING AND PHENOTYPING MIXED POPULATIONS OF HIV INFECTED CELLS

该项目旨在开发和比较极其敏感的 用于检测和分离罕见感染细胞的测定 主要是未感染的细胞背景（例如血液）。 电流诊断 HIV感染的检测依赖于检测循环病毒抗体 或从受感染细胞释放的病毒抗原。 没有 鉴定或定量受感染细胞数量的许可试验 在血液或其他组织中。 有许多研究检测， 然而，病毒或受感染的细胞，它们的敏感性极限不是很好， 确立了习 我们计划开发和表征免疫荧光的灵敏度 用于显微镜的测定和基于核酸扩增的测定 载玻片以及荧光激活细胞分选仪和分析仪 （FACStar和FACScan）。 流式细胞仪的能力允许筛选统计学上 大量的细胞，和排序能力允许无菌，克隆 分离感染细胞。 这些技术将用于1） 评估特定血液亚群内感染细胞的数量，2） 克隆分离病毒的主要毒株，和3）定位 在体外实验中感染。 我们正处于这个项目的早期阶段，已经开始准备， 验证用于灵敏度测定的对照细胞培养物，2）开始 评价DTTD可用的HIV阳性血清池以及HIV 单克隆抗体，3）经过FACSTAR Plus操作培训的人员， 4)制定了血液亚群表型分析的方案。

This project is aimed at developing and comparing extremely sensitive assays for detection and isolation of rare infected cells in a predominantly uninfected cell background (e.g. blood). Current diagnostic tests for HIV infection depend on detection of circulating viral antibodies or virus antigen which is released from infected cells. There are no licensed assays which identify or quantitate the number of infected cells in blood or other tissues. There are numerous research assays which detect virus or infected cells, however, their limits of sensitivity are not well established. We plan to develop and characterize the sensitivity of immune fluorescence assays and nucleic acid amplification based assays for use on microscope slides as well as on the Fluorescence Activated Cell Sorter and Analyzer (FACStar and FACScan). FACS capability allows screening of statistically significant numbers of cells, and sort capability allows sterile, clonal isolation of the infected cells. These techniques will be used to 1) evaluate the number of infected cells within specific blood subsets, 2) clonally isolate primary strains of virus, and 3) localize sites of infection in in vitro experiments. We are in the early stages of this project and have 1) begun preparing and validating control cell cultures for sensitivity determinations, 2) begun evaluation of HIV positive serum pools available at DTTD, as well as HIV monoclonal antibodies, 3) trained personnel for FACSTAR Plus operation and 4) developed protocols for blood subset phenotyping.