HIV-1 P24抗原国家参考品的研制

收集正常人、HIV感染者和疑似感染者血浆以及HIV-1病毒培养裂解液,对其进行HIV抗体、HIV P24抗原、HCV抗体和HBsAg检测,对HIV P 24抗原阳性者进行HIVRNA检测,并对部分样品进行基因分型.以NIBSCP 24抗原标准品的系列稀释样品作为线性灵敏度参考品.经过实验筛选出20份阴性参考品,10份阳性参考品,10份线性灵敏度参考品,2份精密性参考品,共同组成HIV-1 P24抗原国家参考品,经多家不同的试剂进行标定,制定了相应的标准.稳定性研究结果表明,反复冻融三次对该参考品的稳定性没有影响.由此,初步建立了HIV-1 P24抗原国家参考品,该参考品将对HIV-1 P24抗原、HIV抗体/P 24抗原联合检测试剂的质量控制提供重要依据.     

中国HIV阳性参比品库的建立以及HIV不同生物标志物的意义

为建立HIV阳性样品库并分析HIV不同生物标志物的意义,从我国不同地区以及不同人群中收集HIV感染者或可疑感染者血浆,对其进行HIV抗体、抗原、核酸以及基因型的检测,并用WB试剂对其进行抗体的确认检测。结果显示,该样品库共有样品190份,均为HIV阳性,含有我国流行的主要基因型,即B′、BC、AE和B亚型;HIV抗体S/CO值小于10者占11.1%,在10~15之间者占63.2%,大于15的占25.8%;病毒载量在50~103copies/mL者占7.9%,在103~105copies/mL者占82.2%,大于105copies/mL者占10.0%。而且抗体S/CO值大于10者,均为HIV抗体确认阳性,小于10者仅有61.9%为抗体确认阳性,但核酸均大于50copies/mL,而且抗体不确定样品的病毒载量均大于105copies/mL,但抗体不确定的8份样品中仅有4份样品为P24抗原阳性。结果提示该样品库样品来自于HIV感染的不同时期,可用于对HIV的不同试剂进行评价;而且核酸的检测可有助于对HIV早期感染的明确诊断。     

EB病毒衣壳抗原IgM抗体检测试剂参考品的建立

目的 建立EB病毒衣壳抗原IgM抗体检测试剂国家参考品并制定质量标准。方法 收集并筛选EB病毒衣壳抗原IgM抗体阳性和阴性血浆样本,建立EB病毒衣壳抗原IgM抗体检测试剂国家参考品并进行均匀性和稳定性研究,经10个实验室的协助标定,确定参考品的质量标准。结果 建立的EB病毒衣壳抗原IgM抗体检测试剂国家参考品包括阳性参考品6份、阴性参考品10份、重复性参考品1份和检测限参考品3份。参考品均匀性变异系数(coefficient of variation,CV)为3.1%,满足行业标准CV≤15.0%的要求;2～8℃放置7 d、室温放置3 d和反复冻融3次对参考品均无影响。质量标准为：阳性符合率应≥4/6,阴性符合率应为10/10,重复性(2个浓度水平)的检测结果应均为阳性且CV均≤15.0%,检测限参考品L1应为阳性,L2～L3不作要求。结论 建立的EB病毒衣壳抗原IgM抗体检测试剂国家参考品可用于相关试剂研发的质量控制及评价。     

鼠疫耶尔森菌抗原检测试剂用国家参考品的研制

目的研制鼠疫耶尔森菌(Yersinia pestis)抗原检测试剂用国家参考品。方法通过对5株鼠疫耶尔森菌和10株非鼠疫耶尔森菌的菌种检定、培养及灭活、抗原检测,组建鼠疫耶尔森菌抗原检测试剂用国家参考品。对参考品进行样品均匀性以及稳定性评估,组织5家实验室协作标定。结果参考品由5份阳性、10份阴性、1份最低检出量以及1份重复性样品组成,均匀性和稳定性良好。协作标定结果显示:①5份阳性参考品阳性率100%;②10份阴性参考品阴性率100%;③最低检出量参考品的检出量不高于1.0×10~6个菌/mL;④重复性参考品检测反应结果应一致(酶联免疫法、上转发光免疫层析法等CV<5%)。结论建立的鼠疫耶尔森菌抗原检测试剂用国家参考品填补了相关领域的空白,可用于相关试剂的质量控制。     

应用斑点金免疫渗滤试验快速同步检测抗HIV-1，HIV-2 IgG抗体

应用斑点金免疫渗滤试验(dotimmunogoldfiltrationassay,DIGFA)建立了一种同步快速检测四种抗HIV-1/2IgG抗体的HIV诊断试纸。通过基因工程技术在大肠杆菌中表达了5种HIV抗原蛋白片段(P24,GP41,GP36,GP120V3,GP120C)。这5种抗原蛋白首先被固定在硝酸纤维素膜上,然后滴加待测血清,其中的病毒抗体通过免疫反应与抗原结合,再加胶体金标记的葡萄球菌蛋白A(SPA),待其渗过膜片后,洗涤,即可形成肉眼可见的红色斑点。用已确证的21份HIV阳性血清(其中包括1份HIV-1标准阳性血清和1份HIV-2标准阳性血清)和30份阴性血清进行了试验,结果表明该快速检测方法与ELISA方法无显著差异。该检测方法不需任何仪器,仅凭肉眼即可判定结果,整个检测过程不超过5分钟。与传统的的ELISA法相比,具有方便快速,成本低廉,应用范围广等优点。同时,此HIV快速诊断试纸可以同步检测并区分针对HIV-1和HIV-2感染的不同检测标志物(抗P24、GP41、GP120和GP36抗体),这对提高快速检测的灵敏度和准确性,以及对判断HIV感染者是否临近或已进入AIDS期有着较高的应用价值。     

应用斑点金免疫渗滤试验快速同步检测抗HIV-1,HIV-2 IgG抗体

应用斑点金免疫渗滤试验(dotimmunogoldfiltration assay,DIGFA)建立了一种同步快速检测四种抗HIV-1/2IgG抗体的HIV诊断试纸.通过基因工程技术在大肠杆菌中表达了5种HIV抗原蛋白片段(P24,GP41,GP36,GP120V3,GP120C).这5种抗原蛋白首先被固定在硝酸纤维素膜上,然后滴加待测血清,其中的病毒抗体通过免疫反应与抗原结合,再加胶体金标记的葡萄球菌蛋白A(SPA),待其渗过膜片后,洗涤,即可形成肉眼可见的红色斑点.用已确证的21份HIV阳性血清(其中包括1份HIV-1标准阳性血清和1份HIV-2标准阳性血清)和30份阴性血清进行了试验,结果表明该快速检测方法与ELISA方法无显著差异.该检测方法不需任何仪器,仅凭肉眼即可判定结果,整个检测过程不超过5分钟.与传统的的ELISA法相比,具有方便快速,成本低廉,应用范围广等优点.同时,此HIV快速诊断试纸可以同步检测并区分针对HIV-1和HIV-2感染的不同检测标志物(抗P24、GP41、GP120和GP36抗体),这对提高快速检测的灵敏度和准确性,以及对判断HIV感染者是否临近或已进入AIDS期有着较高的应用价值.     

人巨细胞病毒IgM抗体国家参考品的研制

目的为了对人巨细胞病毒(Human cytomegalovirus,HCMV)IgM抗体检测试剂进行统一评价,研制HCMVIgM抗体国家参考品,用于控制试剂盒的质量。方法收集正常人与感染者的标本,采用多实验室联合标定的方法确认参考品的试验结果,并经一系列的破坏条件进行稳定性和均匀性考核。结果考核的敏感性和准确性符合国家参考品的要求。结论该参考品能用于临床检测试剂的质量控制。     

重组HIV-1抗原的应用及评价

将纯化的基因重组HIV-1P24和融合蛋白P24-gp41包被微孔板,用ELISA分别检测正常人血清和HIV-Ab国家参比品（Panel),rP24对20份HIV-Ab阳性Panel的检出率为95%（19/20）,对20份HIV-Ab阳性Panel通过率为90%（18/20）,P24-gp41对20份HIV-Ab阳性Panel检出率为90%（18/20）,对20份HIV-Ab阴性Panel的通过率为60%（12/20）;rP24和P24-gP41对正常人血清的检测结果均为阴性,结果表明研制的P24具有较高的敏感性和特异性,可作为组份抗原用于HIV抗体诊断试剂盒的生产。     

HIV-1 p24蛋白在大肠肝菌中的高效可溶性表达、一步纯化及抗原性分析

合成引物扩增HIV-1 p24基因,并将其克隆到pQE-30质粒中,使其在大肠杆菌E.coli M15中以IPTG诱导高效表达,经SDS-PAGE分析,该表达产物约占菌体总蛋白20%,并且以可溶蛋白的形式存在于细菌裂解液上清之中.经镍离子柱亲和层析一步纯化,洗脱产物中p24蛋白纯度达95%.ELISA分析表明,该蛋白可与HIV感染者血清发生特异性免疫反应.以此蛋白交联Sepharose 4B,亲和层析纯化HIV感染者血清中的抗体,用所得抗体与HIV确认试剂反应,发现该纯化抗体仅与确认试剂中的p24蛋白反应.上述结果表明在大肠杆菌中已经高效表达了可溶性HIV-1 p24蛋白,该蛋白具有良好的抗原性.     

双抗体夹心ELISA检测HIV-1 gp41抗原方法的建立

目的:建立检测HIV-1gp41抗原的双抗体夹心ELISA,并探讨其临床应用的可行性。方法:用饱和硫酸铵(SAS)纯化抗HIV-1gp41-5单克隆抗体(mAb),用HRP标记后建立双抗体夹心ELISA法,对其灵敏度及特异性进行检测,并用该方法对40份HIV-1阳性血清进行了检测。结果:用mAbE12(5μg/mL)为包被抗体,2H6为酶标记抗体(1∶900)建立了双抗体夹心ELISA法,检测gp41-5多肽的灵敏度是100pg/mL。对HIV-1阳性血清中gp41抗原的检出率为67.5%(27/40)。结论:建立了特异性强、灵敏度良好的检测HIV-1gp41抗原的双抗体夹心ELISA法。     

Multi-Centre Evaluation of the Determine HIV Combo Assay when Used for Point of Care Testing in a High Risk Clinic-Based Population

Background

Determine HIV Combo (DHC) is the first point of care assay designed to increase sensitivity in early infection by detecting both HIV antibody and antigen. We conducted a large multi-centre evaluation of DHC performance in Sydney sexual health clinics.

Methods

We compared DHC performance (overall, by test component and in early infection) with conventional laboratory HIV serology (fourth generation screening immunoassay, supplementary HIV antibody, p24 antigen and Western blot tests) when testing gay and bisexual men attending four clinic sites. Early infection was defined as either acute or recent HIV infection acquired within the last six months.

Results

Of 3,190 evaluation specimens, 39 were confirmed as HIV-positive (12 with early infection) and 3,133 were HIV-negative by reference testing. DHC sensitivity was 87.2% overall and 94.4% and 0% for the antibody and antigen components, respectively. Sensitivity in early infection was 66.7% (all DHC antibody reactive) and the DHC antigen component detected none of nine HIV p24 antigen positive specimens. Median HIV RNA was higher in false negative than true positive cases (238,025 vs. 37,591 copies/ml; p = 0.022). Specificity overall was 99.4% with the antigen component contributing to 33% of false positives.

Conclusions

The DHC antibody component detected two thirds of those with early infection, while the DHC antigen component did not enhance performance during point of care HIV testing in a high risk clinic-based population.     

核酸定量检测试验应用于HIV-1感染实验室诊断的探讨

目的:探讨核酸定量检测在HIV-1感染实验室诊断中的应用。方法:选取145例第四代抗原/抗体联合诊断筛查试验为阳性反应的血浆样本,分别用Western印迹和HIV-1核酸定量方法进行检测,综合对比分析2种方法检测结果。结果:Western印迹检出阳性样本120例,不确定样本17例,阴性样本8例;HIV-1核酸定量试验检出结果大于检测限样本131例,其中包括12例Western印迹不确定样本、2例Western印迹阴性样本;有3例Western印迹阳性样本用HIV-1核酸定量检测试验未能检出。结论:核酸定量检测试验对于HIV-1感染阳性样本是一种有效的实验室诊断方法;对HIV-1核酸定量检测结果为"TND"的样本,建议加做Western印迹或结合其他补充试验结果进行综合诊断。     

Continuing transmission of sexually transmitted diseases among patients infected with HIV-1 attending genitourinary medicine clinics in England and Wales.

OBJECTIVE--To determine whether those who are aware of being infected with HIV continue to adopt behaviours that place others at risk of HIV infection. DESIGN--Ongoing survey of current diagnosis of sexually transmitted disease and awareness of HIV infection among patients attending genitourinary medicine clinics. SETTING--Six genitourinary medicine clinics in England and Wales (two in London and four outside) participating in unlinked anonymous HIV serosurveillance during 1990-3. SUBJECTS--All attenders having blood drawn for syphilis serology for the first time during the calendar quarter of attendance. MAIN OUTCOME MEASURES--The proportion of syphilis serology specimens with antibody to HIV-1 detected by unlinked anonymous testing of the residue. The proportion of attenders infected with HIV-1 who remained clinically undetected, and the proportion who had another recently acquired sexually transmitted disease. RESULTS--Of 85441 specimens tested, 2328 (2.7%) were positive for antibodies to HIV-1. About 30% of these specimens were from attenders whose HIV-1 infection remained clinically undetected. HIV-1 infection was found to coexist with another recently acquired sexually transmitted disease in 651 attenders, of whom 522 were homosexual or bisexual men. Of these, 245 (47%) already knew themselves to be infected with HIV-1. This proportion increased between 1990 and 1993. CONCLUSIONS--A considerable proportion of patients infected with HIV-1 are not identified by voluntary confidential HIV testing in genitourinary medicine clinics. Substantial numbers of homosexual or bisexual men attending genitourinary medicine clinics continue to practise unsafe sex despite being aware of their infection with HIV-1.     

免疫荧光检测HIV—1抗体方法的建立和应用

免疫荧光法检测HIV抗体是确认HIV感染的方法之一［l］,与蛋白印迹法相比,它的特异性更高,易于鉴别非特异反应,更为经济、快速、容易操作。为了拥有更多的HIV抗体检测手段,我们用感染和未感染HIV－l的HeLaCD“细胞制备抗原片,建立了免疫荧光检测HIV抗体的方法,并进行了初步应用。材料和方法1细胞和病毒HeLaCD4”细胞,引自美国BruceChesebro博士的实验室,用含10％热灭活小牛血清的RPMI1640培养基培养和传代。HIV－l株引自美国。2血清艾滋诊断试剂国家参比品,批号9602Z16份经蛋白印迹确认的HIV抗体阳性血清;10份健康…     

Application of Polymerase Chain Reaction to Detect HIV-1 DNA in Pools of Dried Blood Spots

Nucleic acid tests that detect HIV infection at an early phase are available and have been applied on individual dried blood spot (DBS). The present study was undertaken with an aim to evaluate the feasibility of performing PCR for HIV-1 DNA on pools of DBS as an alternative to individual testing. Standardization of PCR by a modified Amplicor HIV-1 DNA assay version 1.5 (Roche molecular diagnostics, USA), on pooled DBS was performed using five confirmed HIV reactive samples with known low viral load of HIV-1 and HIV non-reactive samples in pools of 5, 10 and 20 DBS. After successful standardization of pooling procedure, a total of 183 pools (of 10 DBS each) were prepared from 1,823 DBS samples, collected from a population-based study that tested negative for HIV antibodies and p24 antigen. All these pools were screened for HIV-1 DNA by the Amplicor assay. Standardization of pooling procedure indicated that pooling of 10 DBS gave an optimum result. Out of 183 pools tested, one pool of 10 samples was positive and of these ten DBS that were tested individually to identify the positive DBS, one sample was detected to be positive for HIV-1 DNA. Our study demonstrates that PCR for HIV-1 DNA can be successfully performed on pools of DBS. However, this may be needed only on specialized studies of HIV and not for routine epidemiology studies as only a very small fraction of cases would be missed if only antibody/antigen testing were done.     

不同人类免疫缺陷病毒感染途径群体中戊型肝炎病毒的流行情况

本研究旨在了解不同人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染途径群体中戊型肝炎病毒(hepatitis E virus,HEV)抗体情况,探讨HEV疫苗接种的必要性。采集HIV感染者的血清或血浆,利用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测HEV IgG抗体、IgM抗体及抗原,荧光定量聚合酶链反应(polymerase chain reaction,PCR)检测HEV核酸,Roche高纯化HIV-1核酸定量检测试剂盒(PCR荧光法)检测HIV感染者的HIV载量。比较分析不同HIV感染途径群体中HEV流行率的差别。结果显示,HIV感染者中HEV IgG抗体的阳性率为37.4%,静脉吸毒、成分献血和传播途径不明HIV感染群体的HEV IgG抗体阳性率分别为49.3%、39.5%和30.4%。HEV核酸荧光PCR检测结果均为阴性。3种HIV感染群体之间HEV IgG抗体阳性率差异无统计学意义(χ~2=2.978,P0.05)。HEV IgG阳性与阴性感染者之间HIV载量差异无统计学意义(P0.05)。结果提示,为保护HIV感染者免受HEV感染,应考虑接种HEV疫苗。     

HIV感染早期病毒p24蛋白的检测

目的:建立敏感、特异的检测血清中HIV-p24蛋白的方法,作为HIV感染早期即窗口期的监测手段。方法:用纯化的p24蛋白免疫小鼠及家兔,获得单克隆及多克隆抗体,经DEAE-52阴离子交换柱纯化后,标记辣根过氧化物酶,建立ELISA双抗体夹心及间接双抗体夹心方法,检测HIV-p24蛋白。结果:包被单抗、标记多抗或包被多抗、标记单抗,均能特异地检出系列稀释的p24蛋白,包被混合单抗较包被多抗更敏感;经标记的抗种属抗体放大可明显提高检测的敏感性。结论:建立了敏感、特异的检测p24蛋白的双抗体夹心法,间接放大方法可检出50pg/mL的HIV-p24蛋白,检测敏感性与国际同类产品相似。     

The Implementation and Appraisal of a Novel Confirmatory HIV-1 Testing Algorithm in the Microbicides Development Programme 301 Trial (MDP301)

We describe the application of a novel HIV confirmatory testing algorithm to determine the primary efficacy endpoint in a large Phase III microbicide trial. 9385 women were enrolled between 2005 and 2009. Of these women, 537 (6%) had at least one positive HIV rapid test after enrolment. This triggered the use of the algorithm which made use of archived serum and Buffy Coat samples. The overall sample set was >95% complete. 419 (78%) of the rapid test positive samples were confirmed as primary endpoints using a combination of assays for the detection of HIV-specific antibodies (EIA''s and Western Blot), and for components of the virus itself (PCR for the detection of nucleic acids and EIA for p24 antigen). 63 (12%) cases were confirmed as being HIV-positive at screening or enrolment and 55 (10%) were confirmed as HIV negative. The testing algorithm confirmed the endpoint at the same visit as that of the first positive rapid test in 90% of cases and at the time of the preceding visit in 10% of cases. Of the 63 cases which were subsequently confirmed to be HIV-1 positive at or before enrolment, 54 specimens contained no detectable HIV antibodies at screening or enrolment. However, 43 were positive using an EIA which detects both HIV antigen and antibody and also had a positive p24 antigen or HIV PCR test, which was highly suggestive of acute infection. There were 6 unusual cases which had undetectable HIV-1 DNA or RNA. In 4 of the 6 cases the presence of HIV-1-specific antibodies was confirmed by Western Blot. One of these cases with an indeterminate Western Blot was a previous vaccine trial participant. The algorithm served the objectives of the study well and can be recommended for use in determining HIV as an endpoint in clinical trials.

Trial Registration

ISRCTN.org ISRCTN 64716212     

Antigen detection in primary HIV infection

Serial blood samples were obtained from 21 homosexuals who had developed symptomatic primary infection with human immunodeficiency virus (HIV) after a median incubation time of 14 days. During the first two weeks after the onset of illness HIV antigen (p24) was detected in the blood by enzyme linked immunosorbent assay (ELISA). During the second and third weeks after the onset of illness p24 antibody was detected by Western blot assay and antigen concentrations rapidly decreased to undetectable values. Dissociation of antigen-antibody complexes showed complexed antigen during the phase of declining concentrations of free antigen. Neither free nor complexed antigen was detected in any serum samples for several months thereafter, which suggested that failure to detect HIV antigen reflected low or absent synthesis of viral protein rather than masking of antigen by antibodies. Reappearance of HIV antigen with a fall in p24 antibody concentration was observed in a few patients six months or more after the onset of disease.The combined use of antigen and antibody assays made it possible to obtain evidence of infection with HIV in all of the 95 serum samples tested, illustrating the usefulness of these assays for diagnosing infection with HIV in its early stages.