Patiris Peter, Hanson Carl
California Department of Health Services, Richmond, California 94804, USA.
AIDS Res Hum Retroviruses. 2005 Sep;21(9):784-90. doi: 10.1089/aid.2005.21.784.
To accurately quantify HIV-1 neutralizing antibody titers in primary human cells, we developed a single round, focus-forming unit (FFU) reduction assay in human peripheral blood mononuclear cells (PBMC). Infected PBMC were enumerated by a reverse ELISPOT technique in which they were incubated under agarose in the presence of a protease inhibitor in anti-p24 antibody-coated microtiter plates. Viral p24, secreted in the immediate vicinity of infected cells and captured by immobilized antibodies, was subsequently stained using gold-labeled anti-p24-antibody and a precipitating silver substrate. The resulting spots were counted visually, without the aid of a microscope, and percent neutralization titers were determined using curve-fitting software. Results of this ELISPOT neutralization assay (ENA) for 15 HIV-positive human specimens were compared with results from a standard PBMC neutralization assay (standard assay) that measured neutralization as a function of p24 concentration by enzyme immunoassay (EIA). The ENA measures FFU reduction of both syncytium-inducing (SI) and non-syncytium-inducing (NSI) primary isolates. Completed assay plates may be retained as a physical record of results or saved as an image using a flat-bed computer scanner.
为了准确量化原代人细胞中的HIV-1中和抗体滴度，我们开发了一种在人外周血单核细胞（PBMC）中进行的单轮焦点形成单位（FFU）减少试验。通过反向ELISPOT技术对感染的PBMC进行计数，即将它们在抗p24抗体包被的微量滴定板中，于蛋白酶抑制剂存在的情况下在琼脂糖下孵育。在感染细胞紧邻区域分泌并被固定化抗体捕获的病毒p24，随后用金标记的抗p24抗体和沉淀性银底物进行染色。目视计数所得斑点，无需借助显微镜，并使用曲线拟合软件确定中和滴度百分比。将该ELISPOT中和试验（ENA）对15份HIV阳性人类标本的结果与标准PBMC中和试验（标准试验）的结果进行比较，标准试验通过酶免疫测定（EIA）将中和作为p24浓度的函数进行测量。ENA可测量合胞体诱导（SI）和非合胞体诱导（NSI）原代分离株的FFU减少情况。完成的试验板可作为结果的实物记录保留，或使用平板计算机扫描仪保存为图像。
