【作者】 颜浩；

【导师】 蒋岩；

【作者基本信息】 中国疾病预防控制中心 ， 免疫学， 2014， 博士

【摘要】 研究背景用于估算HIV-1新发感染率的实验室检测技术已有十多年的发展,其中大部分方法都是基于血清学的抗体检测。目前普遍使用的BED和亲和力等基于酶联免疫的方法均需要配备相应的实验设备,熟练的实验操作人员,并且需要若干小时得到实验结果。为了解决这些问题,研究一种可快速准确地进行新发感染检测的方法显得很有必要。斑点金免疫渗滤(Dot immunogold filtration assay, DIGFA)技术是胶体金免疫技术与固相膜相结合的一项快速检测技术,因其快速、简便、经济、结果直观等特点而得到广泛的发展和应用。本文尝试建立了基于金标银染免疫渗滤技术的可对样本同时进行HIV-1常规抗体和新发感染快速检测的方法,以期为节省新发感染检测成本、提高检测效率提供可靠的工具。研究目的一、建立可同时对样本进行HW-1常规抗体和新发感染快速检测的方法；二、研究快速方法应用于HIV-1新发感染检测的窗口期参数；三、研究快速方法应用于HIV-1新发感染检测的假近期感染率参数；四、评价快速方法检测HIV-1抗体和新发感染的可行性及适用性,与现有的商品化试剂检测结果进行对比,为开发用于HIV-1新发感染快速检测的有效方法提供技术平台。研究方法一、以HIV-1M组多亚型基因重组gp41抗原(Recombinant immunodominant region of gp41of HIV-1group M, rIDR-M)和普通重组HIV-1gp41抗原作为探针包被硝酸纤维素膜分别用以检测HIV-1新发感染和常规抗体,胶体金标记的抗人IgG作为显色试剂,通过银染进一步增强反应信号,建立快速检测方法并进行初步验证；二、通过检测250份血清学系列阳转样本(62名血清学阳转感染者),利用SPSS17.0软件对汇总数据进行统计分析,获得快速方法应用于新发感染检测的窗口期参数；三、通过对256例已知为长期感染(感染时间大于1年)并且未进行抗逆转录病毒治疗的感染者样本的检测,计算快速方法应用于新发感染检测的假近期感染率参数；四、对比582例样本的新发感染检测结果,比较所建立的快速检测方法与BED方法、限制性抗原亲和力方法的符合率、一致性和相关性；85例样本HIV-1抗体检测结果与WB确认实验的结果进行对比,观察其符合率与一致性。研究结果一.rIDR-M和普通gp41抗原分别以15.3μg/ml和500μg/ml作为包被浓度效果最佳,银染试剂用量为50μ1染色时间为3分钟时反应信号最强,确定两个探针的Cutoff值分别为3.50和4.08；建立的快速方法检测5例已知感染时间的阳性样本和1例阴性样本的判定结果全部正确,批内和批间的重复实验分析探针灰度值的变异系数均小于15%,方法具有较好的重复性。二、取rIDR-M抗原探针的Cutoff-3.50,新发感染快速检测方法的窗口期为189天；抗病毒治疗会对快速方法的检测结果产生一定的影响。三、快速检测方法的假近期感染率为2.73％(7/256,95％CI0.73%-4.73%),CD4+T淋巴细胞计数对快速方法检测结果的影响不大。四、快速方法检测新发感染与BED方法的符合率为92.10%(536/582),一致性和相关性均较好(Kappa=0.65,R2=0.9397),与限制性抗原亲和力方法的符合率为95.36%(555/582),且具有很好的一致性和相关性(Kappa=0.75,R2=0.9549)；快速方法检测HIV-1抗体与WB实验结果完全一致,其阳性符合率和阴性符合率均达到了100%。研究结论一、通过对反应体系的优化、各检测探针Cutoff值的确定及结果判定标准的制定,建立了基于金标银染免疫渗滤技术的可同时对样本进行HIV-1常规抗体和新发感染快速检测的方法。二、初步获得快速方法用于HIV-1新发感染检测的窗口期参数为189天。三、初步获得快速方法用于新发感染检测的假近期感染率参数为2.73%,该方法的假近期感染率高于限制性抗原亲和力方法低于BED方法。四、构建的快速检测方法对样本进行新发感染检测与BED和限制性抗原亲和力方法检测结果相比均具有较好的一致性和相关性,快速方法检测HIV-1抗体与WB实验结果对比具有非常高的符合率,该方法可为HIV-1新发感染的现场检测和实验室初筛提供可靠、高效的工具。更多还原

【Abstract】 BackgroundThe development of laboratory assays for the estimation of recent HTV-1infection has more than ten years, most of the assays are based on serological antibody detection. Nowadays, the commonly used methods such as BED-CEIA and avidity assay require standard immunoassay equipment, highly trained personnel and several hours to complete. In order to solve these problems, it is necessary to develop a rapid and accurate method for detection of HIV-1incidence. Dot immunogold filtration assay (DIGFA) is a rapid detection technology which combines the immunogold technology and the solid-phase membrane. With the characteristics of rapid, convenient, economical and visualized, DIGFA has been widely developed and applied. In this study, a rapid HIV test based on immuno-gold-silver staining filtration assay for simultaneous detection of recent HIV-1infection and HIV-1conventional antibody was established, hoping to provide a reliable tool for saving the cost and improve the efficiency of detection of HIV-1incidence.Objectives1. To establish a rapid test for simultaneous detection of HIV-1conventional antibody and recent infection.2. To obtain the mean duration of recent HIV-1infection of the rapid test for incidence calculation.3. To obtain the false recent rate of the rapid test for incidence calculation.4. To evaluate the feasibility and applicability of the rapid test for detection of HIV-1conventional antibody and recent infection, and compare with the existing commercial assays, for providing a technical platform for development of effective method for rapid detection of HIV-1incidence.Methods1. Nitrocellulose membranes were coated by a multisubtype HIV-1gp41recombinant protein (Recombinant immunodominant region of gp41of HIV-1group M, rIDR-M) and common HIV-1gp41recombinant protein for detecting recent HIV-1infection and conventional antibody, respectively. Gold-labeled anti-human IgG conjugate was used as a detection antibody and silver staining was taken for further enhancing the reaction signal, by which the rapid test was established and preliminary assessed.2. Two hundred and fifty seroconversional specimens from two cohort studies (62seroconvertors) were tested and the data was analyzed using SPSS17.0to obtain the mean duration of recency of the rapid test.3. Two hundred and fifty six long-term infection (diagnosed time longer than one year) and antiretroviral therapy naive specimens were tested to calculate the false recent rate of the rapid test.4. Recent infection testing results of582specimens were compared and the agreement, concordance and correlation of the rapid test with BED-CEIA and LAg-Avidity EIA were evaluated. HIV-1antibody testing results of85specimens were compared and the agreement and concordance of the rapid test with western blot results were observed.Results1. The optimal coating concentration of the rIDR-M and common HIV-1gp41recombinant protein was15.3μg/ml and500μg/ml, respectively. When the dosage of the silver staining reagent was50μl and stained for3minutes, the reaction signal got the strongest. The cutoff value of the two probes was3.50and4.08, respectively. The testing results of5positive specimens with seroconversion date and one negative specimen by the rapid test were all correct. The rapid test showed favourable repeatability with coefficient of variation of each probe less than15%.2. When the cutoff value of the rapid test was3.50, the mean duration of recency of this assay was189days. The antiretroviral therapy could affect the results of the rapid test.3. The false recent rate of the rapid test was2.73%(7/256,95%CI0.73%-4.73%), and CD4+T cell counts could not affect the results of the rapid test.4. There was a92.10%(536/582) agreement of final classification (recent or long-term) with the BED assay (kappa=0.65) and a high correlation between the grey value of the rIDR-M probe with the BED ODn (R2=0.9397). While there was a95.36%(555/582) agreement of final classification with the LAg-Avidity EIA (kappa=0.75) and a high correlation between the grey value of the rIDR-M probe with the LAg-Avidity ODn (R2=0.9549). The HIV-1antibody testing results of the rapid test and western blot were in full accord, and the coincidence rates of both positive and negative specimens were100%.Conclusions1. Through the optimization of reaction system, determination of the cutoff value of each probe and development the interpretative criteria of the testing results, a rapid HIV test based on immuno-gold-silver staining filtration assay for simultaneous detection of HIV-1conventional antibody and recent HIV-1infection was established.2. Preliminary obtain the mean duration of recent HIV-1infection of the rapid test was189days.3. Preliminary obtain the false recent rate of the rapid test was2.73%, higher than the false recent rate of the LAg-Avidity EIA and lower than the false recent rate of the BED assay.4. The concordance and correlation of the rapid test with the BED assay and LAg-Avidity EIA for detection of recent infection was satisfactory. There was a high agreement of HIV-1antibody testing results between the rapid test and western blot test. The method described in this study could be a reliable and high-efficiency tool for the field detection and laboratory screening of recent HIV-1infection.更多还原