丙型肝炎病毒感染实验室诊断进展

据世界卫生组织统计,全球丙型肝炎病毒（HCV）的感染率约为3%,每年新发HCV病例约315万例,而高度慢性化是该病的最大特征。据报道慢性丙型肝炎患者10年内约有20%发展为肝硬化,肝硬化患者每年约3%发展为肝细胞癌,严重威胁着人类的健康。     

丙型肝炎病毒抗体阳性人群中抗-HCV、HCV-cAg与HCV-RNA结果的相关性及其联合检测在临床应用价值的评估

目的探讨丙型肝炎病毒抗体阳性人群中抗-HCV、HCV-cAg与HCV-RNA结果的相关性及其联合检测在临床应用的价值,同时界定雅培I2000全自动化学发光仪检测抗-HCV真阳性95%时标本S/CO值的临界点。方法选取全自动化学发光仪检测的抗-HCV阳性血清样本179例(包括11例灰区标本),利用化学发光法检测HCV-cAg,利用RT-PCR法检测HCV-RNA,采用SPSS16.0软件对抗-HCVS/CO值绘制受试者操作特性(ROC)曲线,得到95%真阳性时抗-HCV结果的临界值(S/CO)。再对抗-HCV、HCV-cAg与HCV-RNA进行相关性分析。结果对179例样本进行抗-HCV、HCV-RNA及HCV-cAg的检测,其中HCV-RNA阳性43例,HCV-cAg阳性42例。在43例HCV-RNA阳性的标本中,抗-HCV的阳性检出率为93.02%,HCV-cAg阳性检出率为95.35%。抗-HCV灵敏度为93.02%,特异性为5.88%;HCV-cAg灵敏度为95.35%,特异性为99.26%。且HCV-cAg及抗-HCV阳性率随着HCV病毒含量升高而增高,RNA病毒含量越高,HCV-cAg及抗-HCV的阳性率也越高,差异具有统计学意义(P<0.05)。HCV-cAg联合抗-HCV检测与HCV-RNA的阳性符合率为100%,高于单独检测HCV-cAg或抗-HCV的阳性符合率95.35%、93.02%。以HCV-RNA检测为金标准,当抗-HCV的S/CO值>4.42时,真阳性率可达到95%。结论HCV-cAg与HCV-RNA检测的阳性符合率高达95.35%,故HCV-cAg可作为丙型肝炎早期诊断的特异性指标。为保证标本真阳性率达到95%,建议使用美国雅培I2000仪器进行丙肝抗体检测时,若抗-HCV结果(S/CO)小于4.42的标本,抗-HCV、HCV-cAg与HCV-RNA联合检测可有效降低丙型肝炎窗口期的漏检率,为丙型肝炎的早期发现、早期诊断、早期治疗提供有力证据。     

探讨三种检测方法在丙型肝炎诊断中的应用价值

目的探讨ELISA法检测丙型肝炎病毒核心抗原(HCVcAg)、病毒抗体(抗-HCV)及RT-PCR法检测丙型肝炎病毒RNA(HCV-RNA)3种方法在丙型肝炎诊断的应用价值。方法采用HCVcAg ELISA试剂盒,抗-HCV ELISA试剂盒及HCV-RNA PCR试剂盒,对临床200例疑似丙肝病毒感染的样本进行HCVcAg、抗-HCV和HCV-RNA检测。结果 HCVcAg阳性检出率为42%;HCV-RNA阳性检出率最高,为61%;抗-HCV阳性检出率为52%。Kappa检验示3种检测方法结果阳性吻合度基本一致。HCVcAg阳性检出率随着HCV病毒含量的升高而升高。结论 3种方法中,RT-PCR检测HCV-RNA仍是判断丙肝感染最准确方法。HCV核心抗原检测可以有效缩短窗口期,联合运用抗-HCV和HCVcAg或抗-HCV和HCV-RNA,能有效降低单独使用抗-HCV检测的漏检风险。HCVcAg可作为HCV抗体常规检测的补充指标,提高检出率。     

合成肽抗原抗丙型肝炎病毒ELISA诊断方法的研究

用人工合成的丙型肝炎病毒（ＨＣＶ）结构区寡肽Ｃｐ－１９及非结构区寡肽ＮＳ４－２１组装成抗－ＨＣＶＥＬＩＳＡ试剂。经中国药品生物制品检定所抗－ＨＣＶ标准系列血清验证二次。第一次参比血清组结果与预期结果完全相符，第二次参比血清组除１份弱阳性标本漏检外，其余也完全相符，但检测血清的稀释度较标准稀释度低。试剂重复性好，在１８～２０℃放置７天，４℃放置３个月，仍保持原活性。试剂与国内合成肽抗－ＨＣＶＥＬＩＳＡ试剂水平相当，可供临床检测之用。     

酶联免疫吸附法同时检测血清中的HIV和HCV抗体

用酶联免疫吸附试验检测病毒抗体 ,多为对单一抗体的检测 ,我们将人类免疫缺陷病毒 (ＨＩＶ 1 2 )和丙型肝炎病毒 (ＨＣＶ)抗原包被于同一载体 ,同时检测这两种病毒的抗体 ,取得了与单测法基本一致的效果。ＨＩＶ 1 2抗原和ＨＣＶ抗原为加拿大Ｙｅｓ生物技术研究有限公司产品 ;ＨＩＶ酶联免疫检测试剂盒批号 96 0 5 15 ,ＨＣＶ酶联免疫诊断试剂盒批号 96 0 72 3,均为厦门新创科技有限公司产品。ＨＩＶ和ＨＣＶ抗体阴、阳性国家参照品由卫生部药品生物制品检定所制备 ,批号分别为 96 0 1、96 0 4。 112 0份血清样品取自山东医科大学附属医院…     

酶联法和免疫印迹法检测血抗卵巢抗体的比较

为比较酶联法（ELISA）和免疫印迹法（IBT）检测人抗卵巢抗体（AOA）的灵敏度和准确性,本文以正常妇女为对照,检测不明原因性不孕症和卵巢早衰（POF）患者血清AOA。结果表明POF组AOA阳性率分别为40％（ELISA）和80％（IBT）,不孕症组AOA阳性率为20％（ELISA）和60％（IBT）。两种方法比较,同组阳性率有显著差异（P<0.01）。IBT方法可区别非致病性和致病性AOA,提高诊断的准确性、实验表明ELISA和IBT均可用于检测AOA以了解卵巢功能降低之免疫性病因,但IBT方法更灵敏,更准确。     

检测丙型肝炎患者NS5区抗体的临床意义探讨

目的检测慢性丙型肝炎患者和１４例ＨＣＶ原发感染后ＮＳ５抗体长达一年半的动态变化，探讨ＮＳ５抗体的临床意义。方法应用重组ＮＳ５抗原，建立ＥＩＡ方法进行检测。结果慢性丙型肝炎患者抗－ＮＳ５抗体阳性率为６０．４８％，ＨＣＶ感染后一月抗－ＮＳ５抗体阳性率为１６．３５％，三月为７５％。结论抗－ＮＳ５抗体无早期诊断价值。抗－ＮＳ５抗体持续阳性者，血清ＡＬＴ多明显升高，抗－ＮＳ５抗体与肝脏疾病活动性相关。丙型肝炎患者中存在抗－Ｃ、抗－ＮＳ３、抗－ＮＳ４抗体阴性，而抗－ＮＳ５抗体单独阳性，表明检测抗－ＮＳ５抗体具有独特的诊断价值     

丙型肝炎实验室诊断研究进展

经过十余年的深入研究,已建立起了较为完整的丙型肝炎病毒（HCV）感染诊断系统,包括血清学诊断,基因诊断,基因分型,基因变异,血清学分型和肝脏组织活检诊断等,现概述如下. 1血清学诊断（抗HCV检测） HCV感染的血清学诊断主要是检测HCV抗体.抗-HCV酶免疫法（EIA）适用于高危人群筛查,也可用于HCV感染者的初筛.但抗-HCV阴转与否不能作为抗病毒疗效的指标.用第三代EIA法检测丙型肝炎患者,其敏感度和特异度可达99%,因此,不需要用重组免疫印迹法（RIBA）验证[1].但一些透析、免疫功能缺陷和自身免疫性疾病患者可出现抗-HCV假阳性,因此,HCV RNA检测有助于确诊这些患者是否合并感染HCV.     

化学发光法和酶联免疫法作为筛选试验测定丙肝抗体的评价

目的比较化学发光法(CLIA)和酶联免疫法(EIA)测抗-HCV对诊断HCV感染的检出率,分析CLIA测定抗-HCV作为HCV感染初筛试验的重要临床意义。方法分别用CLIA、EIA方法检测所有临床标本中的抗-HCV,选取抗-HCV初筛阳性样本进行HCV RNA确认试验。结果 6461例样本中,EIA和CLIA测抗-HCV阳性率分别为2.86%和3.17%;确认试验中,CLIA法抗-HCV阳性与HCV RNA的符合率为97.1%,显著高于EIA组(91.9%)。EIA法测抗-HCV,S:CO>5.0以上,与HCV RNA检测符合率可达100%;弱阳性样本,与HCV RNA符合率为69.7%;CLIA法测抗-HCV,S:CO>2.0以上,与HCV RNA检测符合率可达100%;弱阳性样本,与HCV RNA符合率为92.9%。有31例样本CLIA法抗-HCV和HCV RNA阳性,而EIA法抗-HCV阴性;有4例样本EIA法检测为阳性,而CLIA法和PCR确认试验均为阴性。结论CLIA法测抗-HCV作为HCV感染初筛实验,与传统EIA相比,特异性和阳性预测值更高,有效降低了假阳性率,有利于临床医生对HCV感染患者的早期诊断和治疗。     

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.     

丙型肝炎病毒抗体与核心抗原联合检测的诊断效能研究

目的 探讨丙型肝炎病毒抗体(HCV-Ab)与核心抗原(HCV-cAg)联合检测对丙型肝炎的诊断效能.方法 经重组免疫印迹试验确证的92例阳性标本,81例阴性对照标本,同时进行HCV-Ab和HCV-cAg检测,分析HCV-Ab检测法、HCV-cAg检测法及两者联合检测法的敏感性、特异性和ROC曲线下面积.结果 HCV-Ab检测法敏感性为79.3％,特异性为93.8％;HCV-cAg检测法敏感性为87.0％,特异性为90.1％;HCV-cAg与HCV-Ab联合检测法敏感性为92.4％,特异性为88.9％.三种试验比较,敏感性差异有统计学意义(P＜0.05),特异性差异无统计学意义(P＞0.05).三种方法ROC曲线下面积分别为0.866、0.885和0.906.HCV-cAg与HCV-Ab联合检测法,诊断准确性较高.结论 HCV-cAg与HCV-Ab检测方法的联合应用对于丙型肝炎的诊断效能较高.     

丙型肝炎病毒抗体双抗原夹心法化学发光检测的应用

目的 探讨丙型肝炎病毒抗体双抗原夹心法化学发光检测的应用.方法 利用重组融合HCV抗原分别标记生物素及吖啶酯,建立起双抗原夹心法化学发光检测试剂盒,与间接法同时检测了866例阴性标本,440例阳性标本,3套BBI阳转血清盘.结果 双抗原夹心法与间接法灵敏度分别为100％、100％,特异性分别为99.9％、98.7％,特异性差别1.2％(95％可信区间:0.5％～2.1％),BBI阳转血清相对敏感性系数为-1.33.结论 双抗原夹心法特异性及早期感染检测能力优于间接法,将会成为丙型肝炎病毒抗体检测的主要方法.     

A Novel Antigen Detection Immunoassay for Field Diagnosis of Hepatitis C Virus Infection

Abstract

The limitations of dominant methods-based on the detection of anti-HCV antibodies or HCV viremia currently used for the diagnosis of HCV infection enhance efforts to have a rapid, simple, sensitive, and specific alternative diagnostic approach to detect viral antigens. A highly reactive IgG antibody was raised to HCV-NS4 recombinant antigen. The produced antibody showed no cross-reactivity with the other HCV structural and nonstructural recombinant antigens (C1 + 2, C3 + 4, E2/NS1, NS3, NS5). The well established ELISA technique was adapted to detect the new target HCV-NS4 antigen in serum samples. Extremely high agreement was found between the results of ELISA and qualitative detection of HCV-RNA, using a RT-PCR test as a gold standard for the diagnosis of HCV infection. Based on these encouraging results, a novel enzyme immunoassay; dot-ELISA was developed for rapid (?5 min) and simple qualitative detection of the target HCV antigen in serum. The developed method detected the HCV target antigen in 95% of serum samples from HCV infected individuals, with a specificity of 97% using sera of noninfected individuals in comparison with PCR test. The antigen detection method showed high predictive values of positive (99%) and negative (90%). Moreover, the dot-ELISA could detect the HCV target antigen in sera negative for anti-HCV Abs, but positive for HCV-RNA, and in sera of HCV infected individuals with low viremia, as well as those with high viremia, using quantitative RT-PCR. Accordingly, the developed highly sensitive and specific HCV antigen detection method could be applied for mass screening of HCV infection.     

丙型肝炎病毒抗原检测试剂在临床诊断中的初步应用

本文利用重组丙型肝炎病毒(HCV)多表位复合抗原免疫动物制备抗HCV多克隆抗体,建立HCAgELISA检测方法,对不同的临床样品进行检测,分析不同临床样品HCAg检出率,并与荧光定量PCR结果进行对比。95份HCAb阳性的样品中,51份样品HCAg阳性,阳性率为53.7%;88份重症肝病患者血清中检出HCAg阳性33份,阳性率为37.5%,均明显高于其它样品(P<0.05);73份肝功异常但HCAb阴性血清样品,检出HCAg阳性8份,阳性率为11.0%;HCAg与HCAb之间存在相关性(r=0.5076,P<0.01),HCAg与HCV-RNA符合率为85.7%。HCAg ELISA检测可作为临床HCV诊断的检测指标,为临床早期诊断及治疗预后提供依据。     

用ELISA法检测丙型肝炎病毒IgM抗体试剂盒的研究

市售丙型肝炎检测试剂盒仅能检测丙肝ＩｇＧ抗体，本工作建立的丙型肝炎病毒ＩｇＭ抗体检测方法稳定性强，重复性好，检出率高，具有高度特异性，与甲、乙、丁型肝炎及ＣＭＶ、ＥＢＶ患者无交叉反应。健康人血中不含丙肝ＩｇＭ抗体，我们用该方法检测了１０９例输血后肝炎患者，发现３７例急性肝炎中３６例丙型肝炎病毒ＩｇＭ抗体阳性（９７％），其中１６例患者在起病后４个月内随病情好转而ＩｇＭ抗体阴转，７２例慢性输血后肝炎患者中４３例ＡＬＴ明显异常，４３例中有３６例（８３７％）丙肝ＩｇＭ抗体阳性，而２９例ＡＬＴ正常患者仅有５例阳性，Ｐ＜０．０１，说明慢性肝炎丙肝ＩｇＭ抗体阳性多伴有肝病活动。     

Clinical Utility of a New Automated Hepatitis C Virus Core Antigen Assay for Prediction of Treatment Response in Patients with Chronic Hepatitis C

Hepatitis C virus core antigen (HCV Ag) is a recently developed marker of hepatitis C virus (HCV) infection. We investigated the clinical utility of the new HCV Ag assay for prediction of treatment response in HCV infection. We analyzed serum from 92 patients with HCV infection who had been treated with pegylated interferon and ribavirin. HCV Ag levels were determined at baseline in all enrolled patients and at week 4 in 15 patients. Baseline HCV Ag levels showed good correlations with HCV RNA (r = 0.79, P < 0.001). Mean HCV Ag levels at baseline were significantly lower in patients with a sustained virologic response (SVR) than in those with a non SVR (relapse plus non responder) based on HCV RNA analysis (2.8 log₁₀fmol/L vs. 3.27 log₁₀fmol/L, P = 0.023). Monitoring of the viral kinetics by determination of HCV RNA and HCV Ag levels resulted in similarly shaped curves. Patients with undetectable HCV Ag levels at week 4 had a 92.3% probability of achieving SVR based on HCV RNA assay results. The HCV Ag assay may be used as a supplement for predicting treatment response in HCV infection, but not as an alternative to the HCV RNA assay.     

Total HCV core antigen assay: a new marker of hepatitis C viremia for monitoring the progress of therapy

The ability of the total hepatitis C virus (HCV) core antigen assay was evaluated for monitoring the therapeutic responses of HCV-infected patients treated with interferon. The ability to detect and quantitate an independent structural protein component of HCV, in the presence of circulating antibodies, makes this assay a valuable new tool in diagnosis and treatment monitoring. Measurement of total core antigen showed a strong dynamic correlation with HCV RNA data and may serve as an alternative direct marker of viral infection. In addition, with the advent of additional treatment protocols, a rapid, reliable assay for changes in HCV load may permit more frequent patient assessment and tailoring of the therapeutic regimen.     

Quantitative measurement of hepatitis C virus core antigen is affected by the presence of cryoglobulins

Mixed cryoglobulinaemia is associated strikingly with HCV infection. The aim of this study was to assess whether the adherence to proper methods of collecting samples for cryoglobulin detection was critical or not on virological parameters in hepatitis C virus (HCV) patients. We studied 56 consecutive patients. Blood samples were collected using a conventional method and a blood collection method at 37 degrees C adapted to cryoglobulin detection. HCV core antigen and HCV RNA were measured in sera and cryoglobulins issued from both blood collection methods. In cryoglobulin-positive patients, serum concentrations of HCV core antigen, but not that of HCV RNA, were significantly higher when a conventional method was used, compared to a blood collection method at 37 degrees C (P = 0.001). In the cryoprecipitates, concentration of HCV core antigen was optimum when the blood collection method at 37 degrees C, rather than the conventional method, was applied for cryoglobulin detection (P < 10(-4)). The recovery of HCV core antigen in the cryoprecipitate was improved when cryoglobulins were isolated using the blood collection method at 37 degrees C rather than the conventional method (P < 0.001). HCV parameter measurements and cryoglobulin study should not be performed on the same serum samples due to the potential impact of blood collection methods on results.     

Sm抗原纯化及ELISA检测Sm抗体的初步研究

从小牛胸腺中盐析出来的Sm/RNP提取物经DEAE Bio-Ge1 A层析即为Sm抗原。在PAGE检测时Sm抗原在BSA之前有几条染色带。 血清学研究显示,Sm抗原仅与Sm阳性和Sm/RNP阳性血清发生反应,不与RNP阳性血清发生反应。经Sm抗原吸收后,Sm阳性和Sm/RNP阳性血清与Sm抗原的反应降低至接近正常人水平,而上述血清与RNP抗原的反应设有明显降低。结果表明,Sm与RNP已完全分开。ELISA方法检测Sm抗体有10/18的SLE和2/4MCTD是阳性,在14例RA、硬皮病和其它疾病中为阴性。