国内丙型肝炎病毒抗体酶联免疫法试剂评价参考品体系的建立

目的 建立国内丙型肝炎病毒抗体酶联免疫法(EIA)诊断试剂评价参考品.方法 收集2009-03/2010-10期间来自全国不同省份4833份供血人员血清样品,用国产5种抗HCV EIA试剂和国外2种抗HCV EIA试剂分别进行初筛和复检;用Chiron的RIBA HCV 3.0 SIA、Roche HCV定量试剂对筛选样品进行了确认试验,对确认阳性样品进行HCV基因型检测;选择不同强度的确认阳性、阴性样品、混合阳性系列稀释样品、部分特殊样品等建立抗HCV诊断试剂评价参考品.结果 设立了抗HCV诊断试剂评价参考品,包含30份强、中、弱阳性的抗HCV混合阳性样品,10份单片段抗体阳性确认阳性样品,6份评价灵敏度系列稀释样品,30份抗HCV确认阴性样品.结论 本研究设立的抗HCV评价参考品检测结果可靠,样品涵盖背景丰富,对新产品研发和筛选评价高质量的抗HCV诊断试剂有一定参考价值.     

HIV抗体快速检测试剂的临床评价

目的对注册前的HIV抗体快速检测试剂的质量进行临床评价。方法用不同的评价试剂检测440份健康献血员样品、300份HIV抗体强阳性样品以及264份HIV感染高危人群样品，并分别计算其特异性和敏感性。结果17家注册前试剂检测300份HIV抗体强阳性样品的敏感性为100％；检测440份健康献血员样品的特异性为97．7％-100％；检测264份HIV感染高危人群样品的特异性为59．1％-100％，敏感性为70．3％-95．1％。结论不同试剂之间存在敏感性和特异性的差异，而且HIV感染的高危自然人群来源的样品有助于客观地反应试剂的质量差异。     

丙型肝炎病毒抗体（抗-HCV）诊断试剂盒质检评价分析

目的:对进口、国产2个厂家的6个不同批次试剂盒进行抽检,以保证试剂的质量,预防不合格试剂用于标本检测.方法:采用抽检的不同批次的试剂盒检测国家标准品考核血清盘,30份阳性血清,30份阴性血清和4份灵敏度血清,计算其灵敏度,特异性,相对符合率等指标.结果:进口抗丙型肝炎病毒(HCV)诊断试剂盒的6个不同批次试剂盒的特异性为99.6%,灵敏度为100%,相对符合率为98.33%;国产抗-HCV诊断试剂盒的6个不同批次,有1个批次的特异性为90.00%,灵敏度为86.66%,相对符合率为88.33%;其他5个批次的特异性为100%,灵敏度为96.66%,相对符合率为98.33%.结论:进口试剂所有批次全部合格,国产试剂有1个批次不合格,退回厂家.影响试剂质量的因素是多方面的,经国家有关部门检定合格的试剂,在投入使用之前必须进行抽检,确保检测结果可靠.     

人源抗丙型肝炎病毒噬菌体抗体库的构建、筛选及表达

用常规RT PCR法 ,直接从 5名丙型肝炎患者外周血混合淋巴细胞中扩增抗体重链Fd基因和κ轻链基因 ,构建噬菌体抗体Fab库。对抗体库进行 5轮吸附 洗脱 扩增的亲和选择后 ,以ELISA法鉴定抗HCV噬菌体抗体。 5轮亲和选择使特异性噬菌体抗体得到高度富集 ,抗HCV噬菌体抗体阳性克隆达 96 %。对一阳性克隆在大肠杆菌中表达 ,经ELISA及Westernblot分析鉴定 ,证实成功表达出人源可溶性Fab。对抗体基因VH和VκDNA序列进行测定 ,证实所获基因为抗体可变区基因。抗HCV噬菌体抗体库的构建和人源抗HCV单克隆抗体Fab段的制备 ,为HCV感染的诊断、治疗和发病机制的研究提供有效的分子工具。     

人源抗丙型肝炎病毒噬菌体抗体库的构建及筛选

目的　构建人丙型肝炎病毒 (HCV)特异性噬菌体抗体库 ,制备人源抗HCV单克隆抗体 (mAb)。方法　用常规RT PCR法 ,直接从 5例丙型肝炎患者外周血混合淋巴细胞中 ,扩增抗体重链Fd基因和κ轻链基因 ,与噬菌体载体pComb3连接 ,构建噬菌体抗体Fab库。对抗体库进行 5轮吸附 -洗脱 -扩增的亲和选择后 ,以ELISA法鉴定抗HCV噬菌体抗体。结果　RT PCR可有效地扩增出Fd和κ基因 ,并以此构建成容量为 1 2× 10 7的噬菌体抗体库。经 5轮亲和选择可使特异性噬菌体抗体得到高度富集 ,抗HCV噬菌体抗体阳性克隆达 96 %。结论　抗HCV噬菌体抗体库的构建和人源抗HCVmAb的制备 ,为HCV感染的诊断、治疗和发病机制的研究提供有效的分子工具     

4种国产和3种进口第4代HIV抗原抗体检测试剂的质量评价

目的 对4种国产和3种进口第4代HIV诊断试剂的质量进行评价.方法 利用HIV抗体阴性样品库和核酸阳性样品库、BBI阳转血清盘样品等,对4种国产和3种进口第4代试剂的敏感性和特异性、检测HIV-1早期感染的能力进行分析.结果 7种第4代试剂的敏感性均为100％ (95％ CI:99.86％～100％),且1份HIV-1感染窗口期样品均检测为阳性,其中1种进口试剂“8+”值较大(1.0892),其余6种试剂的“δ+”值均比较小(0.0836 ～0.3003).对阴性样品,7种试剂均存在不同程度的假阳性(特异性为97.80％ ～ 99.60％,“δ-”值为-1.3803 -0.4778).对BBI阳转血清盘样品,国产试剂的阳转血清相对敏感性系数为-0.500～0,2种进口试剂则为-0.600和-0.700.结论 7种试剂均具有较高的敏感性和特异性,第4代HIV试剂用于血液筛查可发现HIV感染窗口期样本,对减少HIV传播的风险有一定的意义.但进口第4代试剂检测HIV-1早期感染的能力强于国产第4代试剂.     

梅毒螺旋体抗体快速试剂的实验室性能评价

目的 使用梅毒螺旋抗体快速试剂国家参考品以及商品化的血清转化盘和不同临床时期的梅毒样本对目前市面主要使用的TP抗体快速检测试剂进行性能评价。方法 选取34批次梅毒螺旋抗体快速试剂对梅毒螺旋抗体快速试剂国家参考品以及商品化的血清转化盘和51份不同临床时期梅毒感染样本进行检测,分析不同试剂的检测性能差异并依据检测结果进行评分。结果 34批次梅毒螺旋抗体快速试剂有5批次产品不符合梅毒螺旋抗体快速试剂国家参考品的性能要求,均为阳性参考品符合率项目;34批次梅毒螺旋抗体快速试剂检测血清转换盘阳转天数最大相差4周时间;评价梅毒螺旋抗体快速试剂对51份临床样本检测阳性率为84.3%～100%。来自14个厂家的16批次试剂评分结果为84.3～99.4分。结论 部分梅毒螺旋抗体快速试剂对早期梅毒感染样本的检出率较低,一方面需要选取合适的评价样本,另一方面需要加强试剂生产的工艺稳定性。     

利用RIBA试验确定HCV抗体检测的cut- off值及比较分析

目的 利用重组蛋白免疫印迹分析(RIBA)实验,制定本实验室所用的日本希森美康公司的HISCL-5000型化学发光免疫分析仪及配套的相关试剂检测丙型肝炎病毒抗体(抗-HCV)的cut-off值。方法 在2015年10月至2016年9月所检测的19 341例患者标本中收集抗-HCV阳性标本80例,进行RIBA确认实验后进行分析,再利用ROC曲线计算出cut-off值。结果 在本室HCV抗体阳性率为0.41%的数据中,RIBA结果可分为3种,其中25例(31.3%)为阴性,18例(22.5%)为不确定,37例(46.2%)为阳性。当截断值为3.95 COI时,灵敏度为81.1%,特异性为95.1%。另外当特异性为100%时,截断值为5.25 COI,灵敏度为73%。结论 希森美康HISCL-5000型化学发光免疫分析仪及配套的相关试剂检测丙型肝炎病毒抗体(抗-HCV)与RIBA结果间呈正相关;当cut-off值为3.95 COI时,是一个适合本实验室的cut-off值。     

甲型肝炎病毒IgM抗体快速检测试剂国家参考品的建立

目的 建立甲型肝炎病毒IgM抗体快速检测试剂国家参考品,制订其质量标准,用于该类试剂的质量评价。方法 通过对200多份单采浆站收集到的血浆进行初筛,筛选出16份备选样本。经多家实验室协助标定,分析标定结果,确定参考品的组成及其质量标准,并对参考品稳定性进行评估。结果 本参考品由10份阴性参考品、1份最低检出限参考品和重复性参考品组成。质量标准为10份阴性参考品符合率（-/-）应为10/10,最低检出限参考品要求1:40稀释时检测阳性,重复性参考品要求1:15稀释时连续检测10次,结果应均为阳性,且显色度均一。稀释血浆应均为HCV、HBV和HIV标志物阴性。参考品在4℃放置3、7 d和反复冻融2次条件下相对比较稳定。结论 成功建立甲型肝炎病毒IgM抗体快速检测试剂国家参考品,可用于该类试剂的质量控制及评价。     

三种促甲状腺素受体抗体检测试剂的比较

目的：评价某国产促甲状腺素受体抗体（TRAb）检测试剂的临床诊断性能,为临床应用提供指导。方法：应用国产TRAb试剂与两种进口同种试剂（德国Roche和英国RSR）同时检测77份临床样本,对比分析三种试剂检测结果的相关性、符合率,并结合历史资料回顾,计算国产试剂对Graves’病的诊断敏感性。结果：两种进口试剂所测TRAb在Graves’病的阳性比例均为13/15,对本文样本阴、阳性分类符合率为89.6%,总体相关系数为0.767（P〈0.001）。国产TRAb试剂在本文Graves’病阳性比例为2/15,在历史回顾病例的阳性比例为5/31;与两种进口试剂比较符合率〈60%、检测浓度相关系数〈0.2（P〉0.1）。结论：两种进口TRAb试剂对Graves’病具有较高的临床诊断性能并相互吻合,某国产TRAb试剂缺乏临床应用价值,建议摒弃。     

HBsAg ELISA试剂盒筛检结果的评估

目的 评估部分国产与进口HBsAg ELISA试剂盒筛查血源的价值.方法 选用部分国产和进口HBsAg ELISA试剂,对中国生物制品检定所120份HBsAg临床科研组合血清样本及随机抽取400份东莞市无偿献血者血清标本为验证样本进行同步平行检测,以中国生物制品检定所组合血清及献血者HBsAg阳性并经中和试验证实者为金标准.结果采用四格表法计算两者对等指标并进行评价.结果 国产HBsAg ELISA试剂和进口HBsAg ELISA试剂的灵敏度分别为85.71%（72/84）和100%（84/84）;特异性分别为100%（436/436）和96.55%（421/436）;尤登指数分别为0.86、0.97;两种试剂重复合格率均为100%.结论 试剂的灵敏度以进口HBsAg ELISA试剂较好,但特异性比国产试剂差,两种试剂的重复性均好,两种试剂联合筛检献血者血样标本可提高临床输血的安全性.     

Evaluation of immunochromatography and commercial enzyme-linked immunosorbent assay for rapid detection of norovirus antigen in stool samples

The efficiency of immunochromatography and commercial enzyme-linked immunosorbent assay (ELISA) kit (Denka Seiken Co. Ltd., Tokyo, Japan) were evaluated for rapid detection of norovirus (NoV) from stool specimens. A total of 503 stool specimens collected from infants and young children who suffered from acute gastroenteritis were tested for NoV by the NoV-immunochromatography kit, Denka ELISA kit, and by a monoplex RT-PCR method. The NoV-immunochromatography revealed 78.9% sensitivity, 96.4% specificity, and 92.4% efficiency with the monoplex RT-PCR method. The Denka ELISA kit had a sensitivity of 90.4%, specificity of 96.4%, and an efficiency level of 95%. The findings indicate that the newly developed NoV-immunochromatography kit provides the specificity equal to that of the Denka ELISA kit, even through the sensitivity of detection was lower. However, the advantage of the NoV-immunochromatography kit is less time consuming and simpler. The data show that both the Denka ELISA and the NoV-immunochromatography kits may be used as an alternative method for screening of NoV in stool samples.     

Evaluation of the Prototype Roche DNA Amplification Kit Incorporating the New SSK145 and SKCC1B Primers in Detection of Human Immunodeficiency Virus Type 1 DNA in Zimbabwe

We assessed the sensitivity and specificity of a newly developed DNA PCR kit (Roche Diagnostic Corporation, Indianapolis, Ind.) that incorporates primers for all the group M viruses for the detection of human immunodeficiency virus (HIV) type 1 (HIV-1) infection in Zimbabwe. A total of 202 whole-blood samples from adults whose HIV status was known were studied. This included 100 HIV-1-positive and 102 HIV-1-negative samples selected on the basis of concordant results obtained with two enzyme-linked immunosorbent assay kits. The prototype Roche DNA PCR assay had a 100% sensitivity for the detection of HIV-1 DNA and a specificity of 100%. We conclude that the new Roche DNA PCR kit is accurate for the detection of HIV DNA in Zimbabwean samples, in which HIV-1 subtype C dominates.     

应用ROC曲线评价化学发光法及酶联免疫法对HCV感染的诊断价值

目的 探讨国内五种主流丙型肝炎病毒抗体(抗-HCV)酶联免疫法(ELISA)试剂与进口化学发光法(CLIA)检测性能的可比性及其对HCV感染的诊断价值.方法 应用CLIA及五种ELISA(编号为A、B、C、D、E),同时检测免疫印迹法(RIBA)确认的抗-HCV阳性、阴性血清样本各68例,应用受试者工作特征曲线(ROC)评价其性能指标.结果 CLIA及A、B、C、D、E五种ELISA试剂ROC曲线下面积(AUC)分别为:0.989、0.784、0.945、0.841、0.890、0.883(P ＜0.05),差异有统计学意义;CLIA的敏感性大于五种ELISA试剂(P＜0.05),差异有统计学意义,差异主要在于CLIA 8＞S/CO值≥1标本的符合率较低;几种试剂间特异性无显著性差异(P=0.75).结论 国内五种主流HCV抗体ELISA试剂与进口CLIA对HCV感染均具有较高的诊断价值,利用CHA高敏感性及ELISA的高特异性联合检测抗-HCV,可提高HCV感染的诊断效率.     

Evaluation of the sensitivity and specificity of a ligase chain reaction test kit for the detection of Chlamydia trachomatis.

The sensitivity and specificity of a newly developed ligase chain reaction (LCR) test kit were examined by the use of highly purified elementary bodies (EBs) and in situ inclusions containing reticulate bodies only. The performance of the LCR kit was compared with a commercially available polymerase chain reaction (PCR) test kit, AMPLICOR Chlamydia trachomatis. The number of EBs and inclusions of C trachomatis, respectively, at the detection limit of both kits were two EBs and one inclusion per assay. Neither kit cross-reacted with C pneumoniae, C psittaci and C pecorum EBs or reticulate bodies.     

Evaluation of HIV ELISA diagnostic kit developed at the Institute of Primate Research, Nairobi, Kenya

The first diagnostic kits utilizing the enzyme-linked immunosorbent assay (ELISA) technique were developed in mid-eighties, and since then, this technique has become an increasingly important tool for screening multiple samples of blood or serum for presence of antibodies to various infectious pathogens, especially human immunodeficiency virus (HIV) in blood banks. However, most of the commercial diagnostic kits currently available in the market are too expensive, hence not easily affordable in most Diagnostic Laboratories. We designed an ELISA kit for diagnosis of HIV and compared it with some of the commercial kits. We used blood samples from the blood bank at the National Public Health Laboratory Services (NPHLS) in Nairobi and from patients referred to the Kenya Medical Research Institute (KEMRI) for HIV screening. Two commercial kits were used, Wellcozyme HIV Recombinant kit and Recombigen (env & gag) HIV-1 EIA kit. Out of 1350 samples tested by Recombigen (env & gag) HIV-1 EIA kit, 419 (31.0% ) were positive while 421 (31.2% ) samples were positive by Wellcozyme HIV Recombinant kit. Our ELISA kit detected a total of 431 positive samples out of 1350 (31.9% ), which was almost identical to the results from the other kits. Our kit was nearly identical in terms of sensitivity and specificity to the other two commercial kits used in this study. Thus our ELISA system, which is much cheaper than the commercial kits currently available in the market, offers a more affordable system for routine HIV tests.     

Advantage of using a home-made ELISA kit for detection of Helicobacter pylori infection over commercially imported kits

PURPOSE: To evaluate a home-made ELISA kit for detection of Helicobacter pylori (Hp) infection and comparison of its immunologic criteria with those of foreign commercial kits. METHODS: A home-made IgG ELISA kit was developed using soluble antigenic fractions of Hp proteins. Confirmed sera were tested and serological criteria were evaluated through assessment of 199 serum samples. RESULTS: The accuracy, sensitivity and specificity values of home-made kit were 92, 92 and 90.4%, respectively. These immunologic criteria for Trinity kit were 95.2, 95.2 and 95% in comparison with IBL kit (91.3, 92.2 and 88.5%), BIOHIT kit (72.4, 41.6 and 94.1%) and HelicoBlot2.1 (94.2, 93.4 and 100%). Kappa agreement assessment demonstrated that two of the imported ELISA kits had fair to moderate agreement with the home-made kit while the other one had a poor agreement value. CONCLUSIONS: Apart from comparable values between the home-made kit and the most efficient imported kit (Trinity) there was significant cost benefit. Therefore, we recommend the home-made kit as a suitable substitution for detection of Hp infection in the Iranian population.     

Double-antigen sandwich ELISA for the detection of anti-hepatitis C virus antibodies

A double-antigen sandwich ELISA was developed a detection of HCV antibodies by a recombinant multi-epitope HCV antigen and a biotin-streptavidin amplification system. Three plasma specimens from 1708 individuals who were suspected previously to be HCV-positive using an HCV antibody diagnostic kit (Chuangxin, Xiamen, China) displayed negative results when using the ELISA. These results were validated by a recombinant immunoblotting assay (two were negative, and one was indeterminate). Among 889 blood specimens donated for clinical evaluation, 246 were positive and 630 were negative using the ELISA. The sensitivity and specificity of the ELISA were 98.7% and 100%, respectively. In 43 donors and 14 patients with chronic hepatitis C, the detectable rates for HCV IgM by both ELISA and the HCV anti-IgM detection reagents (Huimin, Shenyang, China) were 100%, and the detectable rate for HCV IgG using an indirect HCV-antibody detection kit (GWK, Beijing, China) was 98.3%. Thus, the double-antigen sandwich ELISA exhibits strong specificity and sensitivity and has been approved by the China State Food and Drug Administration (SFDA). The performance of the double-antigen sandwich ELISA was similar to the Ortho ELISA 3.0. It did not give false-negative results otherwise IgM was undetectable using an indirect HCV-antibody detection kit. This ELISA provides another method for the detection of HCV antibodies.     

Evaluation of two commercial enzyme immunoassays for diagnosis of hepatitis C in the conditions of a virology laboratory

BACKGROUND: Most studies which evaluate antibody detection assays are conducted on blood donors specimens, i.e healthy individuals. Sera collected in patients, vs healthy individuals, can make serological tests difficult because of possible non specific reactions interfering with serological tests. The aim of this work was to compare the specificity and the sensitivity of two commercial automated assays for the detection of hepatitis C virus antibody, Monolisa anti-HCV Plus on the Evolis automate (Biorad) and Axsym anti-HCV 3.0 (Abbott). PATIENTS AND METHOD: The prospective study of specificity included 2020 routine serum samples sent to our virology laboratory. The sensitivity was established with eight commercially available HCV seroconversion panels. RESULTS: The Monolisa and the Axsym assays showed a specificity of 99.64 and 99.12%, respectively. Of 49 specimens from eight commercially available HCV seroconversion panels, the number of positive results was 21 and 24 for the two tests, respectively. CONCLUSION: A statistical analysis of specificity and sensitivity results proved no significant difference between the two tests. Nevertheless, the Monolisa kits could be preferred for its more homogeneous sensitivity than the Axsym test and for its apparent better specificity. The final choice of a kit should also take into account the easiness to perform and an optimal integration in the usual practice of the concerned laboratory.     

Rapid presumptive identification of streptococci directly from blood cultures by serologic tests and the L-pyrrolidonyl-beta-naphthylamide reaction.

The value of the Strepslide kit for the rapid presumptive identification of streptococci directly from blood cultures without prior determination of hemolysis patterns was assessed and compared with that of the Streptex and Phadebact streptococcus kits. Studies involved 94 simulated and 60 clinical isolates of 83 streptococci. The Streptex and Strepslide kits had excellent sensitivity and specificity for group A, B, F, and G organisms, and the Phadebact kit had excellent sensitivity and specificity for groups B and G. Group C reactions usually occurred with all of the streptococcus kits with pneumococci and occasionally with alpha-hemolytic streptococci. Although these kits were unacceptable for group C and D organisms, enterococci which were common clinical isolates could be directly identified in blood cultures by a supplementary rapid L-pyrrolidonyl-beta-naphthylamide biochemical test. Direct application of the Phadebact pneumococcus kit to blood cultures was also assessed with 29 isolates of 20 organisms. The specificity was good, but the sensitivity was only 65.5%.