摘要
目的本研究旨在建立基于酶联免疫斑点试验(ELISPOT)的HIV感染T细胞检测方法,从细胞免疫层面辅助诊断HIV感染。方法使用HIV感染者及健康人外周血单个核细胞(PBMC),系统确定HIV特异性ELISPOT检测方法的核心实验条件,包括:包被抗体与检测抗体的浓度,刺激肽组成与合适浓度,细胞浓度,培养基中白细胞介素-2(IL-2)浓度,并评估细胞样本中去除死细胞对ELISPOT检测结果的影响。将建立的检测方法应用于健康人和HIV感染者样本。结果确定HIV ELISPOT免疫检测方法的最佳条件为:5μg/mL的包被抗体浓度,2μg/mL的检测抗体浓度;刺激肽混合使用终浓度为5μg/mL的Gag、Pol、Env、Nef蛋白表位肽;细胞浓度为2×10^(5)PBMCs/孔;培养基IL-2浓度为20 U/mL。去除死细胞能够提高细胞活率,提高ELISPOT检测斑点数。将检测条件优化后的方法应用于37例HIV感染者和40例健康人,结果与预期相符,检测灵敏度和特异性均达到100%。结论成功建立HIV ELISPOT检测方法,该方法可区分健康人与HIV感染者,弥补目前HIV感染检测缺乏细胞免疫检测方法的不足,为HIV感染的辅助诊断提供新的检测工具。
Objective We aimed to establish a method for the detection of human immunodeficiency virus(HIV)-infected T cells based on an enzyme-linked immunospot assay(ELISPOT)to assist in the diagnosis of HIV infection at the cellular immune level.Methods Peripheral blood mononuclear cells(PBMC)from HIV-infected patients and healthy people were used to systematically determine the core experimental conditions of the HIV-specific ELISPOT detection method,including the concentrations of the coating and detection antibodies,composition and concentration of stimulating peptides,cell concentration,and concentration of interleukin-2(IL-2)in the culture medium.Dead cells were removed from the samples to evaluate their effect on ELISPOT assay results.The established detection method was applied to samples from healthy individuals and HIV-infected patients.Results The optimal conditions for the HIV ELISPOT immune detection method were determined as follows:coating and detection antibody concentrations of 5 and 2μg/mL,respectively;a stimulation peptide mixture containing 5μg/mL each of Gag,Pol,Env,and Nef epitope peptides;a cell concentration of 2x10^(5) PBMCs/well,and an IL-2 concentration of 20 U/mL in the culture medium.The removal of dead cells significantly increased cell viability and the number of spots detected using ELISPOT.When the optimized detection method was applied to 37 HIVinfected patients and 40 healthy individuals,the results wereconsistentwithexpectations,with 100%sensitivityand specificity.Conclusions An HIV ELISPOT detection method was successfully established.This method can distinguish between healthy people and HIV-infected patients,compensating for the lack of cellular immune detection methods for current HIV infection detection and provides a new detection tool for the auxiliary diagnosis of HIV infection.
作者
刘芳芳
赵志艳
王美琳
刘佩
金聪
LIU Fangfang;ZHAO Zhiyan;WANG Meilin;LIU Pei;JIN Cong(National HIV Reference Laboratory,National Center for AIDS/STD Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,Bejing 102206,China)
出处
《中国艾滋病性病》
北大核心
2025年第5期469-474,共6页
Chinese Journal of Aids & STD
基金
国家重点研发计划项目(2024YFC2309503)
传染病溯源预警与智能决策全国重点实验室项目
北京市自然科学基金-昌平创新联合基金资助项目(L234054)。
