一种用于测量HIV-1感染个体中抗体依赖性细胞介导的细胞毒性（ADCC）活性的新型增强型绿色荧光蛋白（EGFP）标记的CEM-NKr流式细胞术方法。

A novel EGFP-CEM-NKr flow cytometric method for measuring antibody dependent cell mediated-cytotoxicity (ADCC) activity in HIV-1 infected individuals.

作者信息

Kantakamalakul Wannee, Pattanapanyasat Kovit, Jongrakthaitae Surat, Assawadarachai Vatcharain, Ampol Silawun, Sutthent Ruengpung

机构信息

Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.

出版信息

J Immunol Methods. 2006 Aug 31;315(1-2):1-10. doi: 10.1016/j.jim.2006.06.005. Epub 2006 Jul 17.

DOI:10.1016/j.jim.2006.06.005

PMID:16884734

Abstract

Enhanced green fluorescent protein (EGFP) was stably expressed in CEM-NKr cell, a natural killer (NK) resistant human T-lymphoblastoid cell line, as EGFP-CEM-NKr cells. The cells pulsed with HIV-1 gp120 were then used as target cells for the measurement of antibody dependent cell mediated-cytotoxicity (ADCC) by flow cytometry. Compromised EGFP-CEM-NKr target cells stained with propidium iodide (PI) showed dual (green-red) fluorescent. Kinetic studies demonstrated that the sum of ADCC activity measured at 1-h and again at 2-h incubations by this flow cytometric method was comparable to the activity at 6 h by the standard chromium (51Cr) release assay (CRA). ADCC activity of HIV-1 seropositive sera measured by this new technique correlated strongly with that of CRA (Pearson's correlation coefficient of 0.832; p-value < 0.001 and intraclass correlation coefficient of 0.903; p-value < 0.001). The EGFP-CEM-NKr stable cell line provides a novel method to measure ADCC activity to HIV-1 gp120 by flow cytometry without pre-staining or pre-labeling target cells.

摘要

增强型绿色荧光蛋白（EGFP）在CEM-NKr细胞（一种对自然杀伤（NK）有抗性的人T淋巴母细胞系）中稳定表达，形成EGFP-CEM-NKr细胞。然后，用HIV-1 gp120脉冲处理的细胞用作靶细胞，通过流式细胞术测量抗体依赖性细胞介导的细胞毒性（ADCC）。用碘化丙啶（PI）染色的受损EGFP-CEM-NKr靶细胞呈现双荧光（绿色-红色）。动力学研究表明，通过这种流式细胞术方法在1小时和2小时孵育时测量的ADCC活性总和与通过标准铬（51Cr）释放试验（CRA）在6小时时的活性相当。通过这种新技术测量的HIV-1血清阳性血清的ADCC活性与CRA的活性密切相关（Pearson相关系数为0.832；p值<0.001，组内相关系数为0.903；p值<0.001）。EGFP-CEM-NKr稳定细胞系提供了一种新方法，可通过流式细胞术在不预先染色或预先标记靶细胞的情况下测量针对HIV-1 gp120的ADCC活性。