Heynen C A, Holzer T J
Abbott Laboratories, Department of Experimental Biology Research, North Chicago, IL 60064.
J Immunol Methods. 1992 Jul 31;152(1):25-33. doi: 10.1016/0022-1759(92)90085-8.
Using flow cytometry, monoclonal antibodies to the HIV proteins p24, gp41 and p17 were evaluated for their ability to detect HIV antigens associated with HIV-infected T cells. Mixtures containing varying ratios of HIV-infected and uninfected cells were subjected to analysis with these monoclonal antibodies. In most cases, the monoclonal antibodies identified the correct ratio of HIV-infected cells to uninfected cells in the mixtures tested. An HIV anti-p24 monoclonal antibody was selected for further studies. Flow cytometric analysis was performed on various populations of cells including uninfected, acutely infected and chronically infected cells. Based on cell population fluorescence intensity three distinct regions were identified. In the first region were cells having low level fluorescence that were considered negative for HIV antigens, a profile detected in uninfected cells, and in the majority of cells in the first days following acute HIV infection. In the second region were those cells exhibiting strong fluorescence such as chronically infected cells or acutely infected cells several days after infection. A third region was identified containing cells that were intermediate in fluorescence intensity. Cells exhibiting intermediate intensity fluorescence appeared to have low concentrations of HIV p24 antigen associated with them either through viral adsorption and uptake or through low level virus expression. These intermediate region cells appeared in the early stages following acute infection, and also when chronically infected cells and uninfected cells were permeabilized together, suggesting a 'leaching' of HIV proteins from highly infected cells to uninfected cells. This leaching type of phenomenon could present problems in determining gating parameters for positive cells since uninfected cells that have associated HIV antigens exhibit higher fluorescence intensity than uninfected cells.
利用流式细胞术，对针对HIV蛋白p24、gp41和p17的单克隆抗体检测与HIV感染T细胞相关的HIV抗原的能力进行了评估。用这些单克隆抗体对含有不同比例HIV感染细胞和未感染细胞的混合物进行分析。在大多数情况下，单克隆抗体能够确定所测试混合物中HIV感染细胞与未感染细胞的正确比例。选择了一种HIV抗p24单克隆抗体进行进一步研究。对包括未感染细胞、急性感染细胞和慢性感染细胞在内的各种细胞群体进行了流式细胞术分析。根据细胞群体荧光强度确定了三个不同区域。第一个区域是荧光水平较低的细胞，被认为HIV抗原呈阴性，未感染细胞以及急性HIV感染后最初几天的大多数细胞呈现这种特征。第二个区域是那些荧光较强的细胞，如慢性感染细胞或感染后数天的急性感染细胞。第三个区域的细胞荧光强度处于中间水平。荧光强度处于中间水平的细胞似乎通过病毒吸附和摄取或低水平病毒表达而与低浓度的HIV p24抗原相关。这些处于中间区域的细胞出现在急性感染后的早期阶段，以及慢性感染细胞和未感染细胞一起通透处理时，这表明HIV蛋白从高度感染细胞“渗漏”到未感染细胞。这种渗漏现象在确定阳性细胞的门控参数时可能会带来问题，因为与HIV抗原相关的未感染细胞比未感染细胞表现出更高的荧光强度。
