部分ELISA抗-HCV阴性和阳性血清丙肝抗体分片段检测分析

目的　探讨ELISA抗 HCV检测的血液丙肝不同基因抗体的反应性和血液HCV感染的具体情况。方法留取本站抗 HCV阴性血清 1 6 8份、阳性血清 80份、可疑血清 6 2份 ,分别进行丙肝分片段检测 ,检测阳性的血清再进行HCVRT PCR检测。结果　 1 6 8份阴性血清、80份阳性血清及 6 2份可疑血清中不同基因抗体的阳性数分别为 3份、76份和 2 4份 ,HCVRT PCR的阳性结果分别为 0份、5 7份和 1 1份 ,两个以上片段检测结果阳性的血清与RT PCR结果高度符合 ,单独C区和NS3阳性的血清仍有一半以上RT PCR结果阳性 ,1例NS5阳性的血清RT PCR检测阳性。结论　HCV C和NS3抗体是抗 HCV阳性的主要血清标志 ,但NS4和NS5抗体在抗 HCV检测中也具有重要意义     

丙型肝炎病毒抗体单片段检测与总抗体检测的比较分析

目的通过对国产HCV抗体分片段酶联免疫检测试剂与进口第三代抗HCV检测试剂检测丙型肝炎病毒抗体的研究来了解丙肝病毒抗体单片段检测的意义。方法采用国产基因重组表达的丙型肝炎病毒不同区(C、NS3、NS4、NS5)特异性抗原分别包被酶标板,以间接EIA法检测75例丙肝患者血清中不同区特异性抗体,并以Abbott公司第三代抗HCV试剂进行总抗体检测,同时做HCVRNA检测。结果75例血清中HCV不同区抗原片段NS3、HCVC、NS4、NS5的抗体检出率分别为933%(7075)、920%(6975)、707%(5375)、640%(4875),含两个片段以上的抗体检出率为947%(7175);抗HCV总抗体的检出率为987%(7475);HCVRNA检出率为774%(5875)。结论HCV不同区抗原片段的抗体检出率有差别,NS3、HCVC检出率较高,其次为NS4、NS5。两种试剂有较好的相关性,HCV抗体单片段试剂可作为检测丙型肝炎病毒抗体的补充试剂,单独片段抗体阳性具有一定意义,动态观察NS3、NS4抗体水平可预测IFN的治疗效果。     

HCV单片段抗原EIA与常规EIA检测丙型肝炎病毒抗体的比较

目的比较国产重组HCV单片段抗原酶免疫(EIA)检测试剂与国产第三代HCV常规抗体(EIA)检测试剂的敏感性和特异性。方法利用基因工程技术重组表达的HCV单片段(HCV-C、NS、NS4、NS)EIA检测试剂和常规EIA试剂检测国家第三代抗-HCV血清考核盘(40份阳性和40份阴性血清样品)及20份不符血样，对检测结果进行统计分析。结果HCV单片段EIA检测试剂总符合率100％，常规试剂为96．2％。结论HCV抗体单片段试剂可作为对HCV常规EIA试剂检测不符血样的确认试剂。     

抗-HCV分片段试剂在辅助诊断HCV感染中的应用

目的探讨抗HCV分片段试剂在辅助诊断HCV感染中的效果。方法收集常规初复检抗HCV不符的临界样本31份,用抗HCV分片段酶联免疫检测试剂进一步检测抗HCV C、NS3、NS4、NS5、膜高变区1(HVR1)抗体。结果31份初复检不符临界样本分片段检测结果为4份阳性(12.90%),10份不确定,即仅1个抗原片段阳性(32.26%),17份阴性(54.84%)。结论对抗HCV初、复检结果不符的临界样本,用抗HCV分片段酶联免疫检测试剂进一步作辅助检测,可降低假阳性,提高检测的特异性,具有一定的临床意义。     

献血人群中丙型肝炎病毒第1高变区抗体检测及意义

目的研究丙型肝炎病毒(HCV)第一高变区(HVR1)抗体检测在献血者血液筛查中的意义。方法采用融合F4HVR1抗原检测不同血清样本中的抗-HVR1的存在情况,并与现有C、NS3、NS4、NS5抗体进行比较。结果在HCV-RNA阳性样本中,HVR1抗体的阳性率为96.8%;在90份可疑HCV感染血清中HVR1抗体的阳性率为61.1%,与C区、NS3区接近,高于NS4区、NS5区(P<0.05),共检测出4份单独HVR1抗体阳性血清。结论在现有HCV诊断试剂基础上,对献血人群进行HCV-HVR1抗体的检测可以提高血液筛查灵敏度,减少HCV的经血传播。     

蛋白芯片检测丙型肝炎病毒分片段抗体

目的用蛋白芯片的方法检测丙型肝炎病毒(HCV)分片段抗体。方法利用生物芯片技术和化学发光技术将高度纯化的抗原HCV Core、NS3、NS4、NS5以特定微阵列固定在固相载体上,用化学发光免疫法检测抗HCV Core、抗HCV NS3、抗HCV NS4和抗HCV NS5。结果174份血清中HCV抗体阳性87份,HCV抗体阴性87份,其中献血员33份,非丙型肝炎的其他肝病患者39份,非肝病者15份。87例雅培公司的AxSYM(微粒子发光)检测抗HCV阳性患者中,蛋白芯片检出阳性85份,阳性率为97.70%;在174份标本中,蛋白芯片检出阳性标本88份,其中抗HCV阳性87份,阳性率98.86%;抗HCV NS3检出49份,阳性率为55.68%;抗HCV NS4检出68份,阳性率为77.27%;抗HCV NS5检出37份,阳性率为42.05%;抗HCV Core检出69份,阳性率为78.41%。结论蛋白芯片显示较高的敏感性和特异性,具有临床实用价值。     

丙型肝炎病毒非结构区3（NS3）I、Ⅱ型血清反应性的比较研究

目的：研究丙型肝炎病毒（HCV）非结构区3（NS3）I型、Ⅱ型血清反应性。方法：利用表达载体PBVIL1对HCV全长NS3区I型、Ⅱ型抗原表位进行克隆表达，经纯化获得电泳纯的抗原。经酶联免疫吸附（ELISA）的方法检测丙型肝炎179份总抗体可疑阳性血清，再用NS3区I、Ⅱ型抗原进行相应抗体检测。结果：通过测定HCV－NS3区I型、Ⅱ型抗体，179份HCV－NS3区I型阳性检出105份，HCV－NS3区Ⅱ型阳性检出95例。结论：重组表达的HCV－NS3 I型、Ⅱ型抗原对丙型肝炎病毒血清反应略有不同。二者具有一定的互补性。     

两种抗HCV试剂检测献血者结果不符追踪观察

目的探讨两种抗HCV试剂检测结果不符标本在抗HCV分片段试剂不同区抗原的反应性,随访和跟踪该类人员在抗HCV和抗HCV分片段试剂中的反应变化.方法利用抗HCV分片段试剂对国家抗HCV参考品及抗HCV试剂检测不符的标本进行测试,并对可跟踪的献血者在6个月后再进行抗HCV及抗HCV分片段试剂检测.结果国家抗HCV参考品中,29份阳性标本均出现二片段或二片段以上阳性,阴性标本全部阴性;103份抗HCV试剂结果不符标本中有6例为二片段阳性,26例为单片段阳性,以NS3、C区为主;6个月后对可以跟踪的86例献血员进行检测,37例两种抗HCV试剂转阴,16例两种试剂均转为阳性,6例抗HCV分片段阴性者己转为一个或二个片段阳性.结论对抗HCV试剂阳性结果不符标本同时应用HCV分片段试剂检测有利于降低HCV阳性血清漏检率,跟踪和复检有助于了解阳性结果不符标本在抗HCV及抗HCV分片段试剂中的反应变化,排除假阳性.     

两种抗HCV试剂检测献血者结果不符追踪观察

目的 探讨两种抗HCV试剂检测结果不符标本在抗HCV分片段试剂不同区抗原的反应性，随访和跟踪该类人员在抗HCV和抗HCV分片段试剂中的反应变化。方法 利用抗HCV分片段试剂对国家抗HCV参考品及抗HCV试剂检测不符的标本进行测试，并对可跟踪的献血者在6个月后再进行抗HCV及抗HCV分片段试剂检测。结果 国家抗HCV参考品中，29份阳性标本均出现二片段或二片段以上阳性，阴性标本全部阴性；103份抗HCV试剂结果不符标本中有6例为二片段阳性，26例为单片段阳性，以NS3、C区为主；6个月后对可以跟踪的86例献血员进行检测，37例两种抗HCV试剂转阴，16例两种试剂均转为阳性，6例抗HCV分片段阴性者己转为一个或二个片段阳性。结论 对抗HCV试剂阳性结果不符标本同时应用HCV分片段试剂检测有利于降低HCV阳性血清漏检率，跟踪和复检有助于了解阳性结果不符标本在抗HCV及抗HCV分片段试剂中的反应变化，排除假阳性。     

蛋白芯片检测丙型肝炎病毒分片段抗体

目的 用蛋白芯片的方法检测丙型肝炎病毒（HCV）分片段抗体。方法 利用生物芯片技术和化学发光技术将高度纯化的抗原HCV Core、NS3、NS4、NS5以特定微阵列固定在固相载体上,用化学发光免疫法检测抗HCV Core、抗HCV NS3、抗HCV NS4和抗HCV NS5。结果 174份血清中HCV抗体阳性87份,HCV抗体阴性87份,其中献血员33份,非丙型肝炎的其他肝病患者39份,非肝病者15份。87例雅培公司的AxSYM（微粒子发光）检测抗HCV阳性患者中,蛋白芯片检出阳性85份,阳性率为97．70％;在174份标本中,蛋白芯片检出阳性标本88份,其中抗HCV阳性87份,阳性率98．86％;抗HCV NS3检出49份,阳性率为55．68％;抗HCV NS4检出68份,阳性率为77．27％;抗HCV NS5检出37份,阳性率为42．05％;抗HCV Core检出69份,阳性率为78．41％。结论 蛋白芯片显示较高的敏感性和特异性,具有临床实用价值。     

生物素化HCV多抗原表位融合基因的克隆及可溶性表达

为了建立丙型肝炎病毒抗体(HCV-Ab)的双抗原夹心ELISA检测方法．克隆表达带有生物素标签的Hcv多种抗原优势表位融合蛋白，选取HCV各抗原如Core，NS3，NS4，NS5和E的优势抗原表位片段编码序列．克隆重组为融合基因，插入Pinpoint^TM Xa-1 T载体中诱导表达，并用Western blot进行抗原性及标签生物素活性鉴定；表达抗原经Promega SoftLink Soft Release Avidin Resin系统亲和层析纯化后包被酶联板，用抗HCV单片段抗体阳性血清对表达融合蛋白各区的抗原性作间接ELISA法鉴定。结果显示：成功构建了带有生物素标签的HCV多抗原表位融合基因表达载体，该载体可在JM109(DE3)中可溶性表达目的蛋白，表达产物携带生物素标签，融合的各片段区均具有良好的抗原性。结论：所构建融合抗原可可溶性表达，可以用作双抗原夹心ELISA的酶标抗原．所含生物素标签也可用作酶联检测的生物素一亲和素信号放大系统。     

The hepatitis C virus genotype and subtype frequency in hepatitis C virus RNA-positive, hepatitis C virus antibody-negative blood donors identified in the nucleic acid test screening program in Poland

BACKGROUND: Since 2002, blood donors in Poland have been tested not only for hepatitis C virus antibodies (anti-HCV) but also for HCV RNA or HCV core antigen. This screening program identifies asymptomatic, recently infected individuals with no anti-HCV (in the "window period"). The aim of this study was to compare HCV genotype and subtype distribution in window-period (wp) donors, anti-HCV-positive donors, and chronic hepatitis C (CHC) patients. STUDY DESIGN AND METHODS: A total of 2.37 million donors were investigated for HCV RNA, and 340,000 for HCV core antigen. HCV genotypes and subtypes were investigated in 50 HCV RNA-positive, anti-HCV-negative donors; in 70 anti-HCV-positive donors; and in 170 CHC patients. Re-questioning of wp donors for probable risk factors was introduced. RESULTS: HCV RNA was detected in 50 donors of 2.71 million (1:54,200) anti-HCV-negative blood donations. Of these 50 donors, 36 percent exhibited Subtype 1b, whereas Subtypes 3a and 4c/d were identified in 40 and 14 percent, respectively. In anti-HCV-positive donors and CHC patients, the frequency of Subtype 1b was significantly higher (75.7 and 85.3%, respectively); in both groups the lower frequency of Subtypes 3a (14.3 and 10.6%, respectively) and 4c/d (4.3 and 1.2%, respectively) was found. The probable source of infection was identified in 9 wp donors. CONCLUSIONS: The frequency of wp donors is 18.5 per 1 million. The unexpected high frequency of Genotype 4 and Subtype 3a and the low frequency of Subtype 1b was observed in wp donors compared to anti-HCV-positive individuals. Additional epidemiologic questioning introduced after HCV RNA detection may help to identify infection source.     

Diagnostic significance of antibody detection to various hepatitis C virus antigens in patients with acute and chronic HCV-infection

AIM: To examine diagnostic value of antibodies to various HCV antigens in patients with acute and chronic HCV-infection. MATERIAL AND METHODS: Enzyme immunoassay has tested blood sera from 136 patients with icteric acute hepatitis C (AHC) and 45 patients with chronic HCV infection for IgG antibodies to antigens of proteins core, NS4, NS5, HCV. Synthetic peptides core-16, NS4-20, NS5-23 were used as antigens. RESULTS: Patients with icteric AHC had IgG antibodies to antigens of both structural protein core and non-structural proteins NS4, NS5 of HCV as early as the first 10 days of jaundice. Occurrence of anti-core and anti-NS4 increases with the disease duration. Incidence of anti-NS4 correlated with duration of previous intravenous drug addiction. In patients with AHC early in the icteric period anti-core, anti-NS4, anti-NS5 were present less frequently than in patients with chronic HCV infection having elevated levels of AlAT. Significant differences were found neither with the group with normal AlAt nor in the spectrum of the detected antibodies between patients with acute and chronic HCV infection. CONCLUSION: Despite different frequency of anti-core, anti-NS4, anti-NS5 detection in patients with icteric AHC and patients with chronic HCV-infection and high AlAT, their high incidence rate in this or that group and absence of differences by the spectrum of the studied antibodies do not allow the fact of their detection to be a diagnostic marker differentiating acute HCV-infection with chronic one.     

丙型肝炎病毒抗体双抗原夹心法酶联免疫试剂的初步临床评价

目的对研制的丙型肝炎病毒抗体（双抗原夹心）酶联免疫检测试剂进行临床评价。方法以克隆表达的HCV多表位嵌合蛋白作为包被抗原,经多表位嵌合蛋白修饰后用于标记抗原,研制HCV双抗原夹心酶联免疫检测试剂;检测血液筛查阴性标本440份,乙肝表面抗原阳性标本90份,HIV抗体阳性标本10份,梅毒螺旋体抗体阳性标本90份及丙型肝炎病毒抗体阳性标本259份。结果对259份HCV抗体阳性标本进行检测时,有3份标本未检出,应用Chiron RIBA确认试剂检测后,结果为阳性。其余标本的检测结果均为阴性。结论采用HCV双抗原夹心酶联免疫检测试剂共检测889份标本,其中只有3份标本结果不一致,总符合率99．7％,敏感性和特异性较好。     

Immunologic events during the incubation period of hepatitis C virus infection: the role of antibodies to E2 glycoprotein. Multicentre Hemodialysis Cohort Study on Viral Hepatitis

BACKGROUND: The study of the sensitivity of screening assays is greatly facilitated by testing the sequential changes in seroconverting individuals. The aim of this study was to investigate the early immunologic response after hepatitis C virus (HCV) infection and to evaluate whether HCV envelope (E2) recombinant antigen would provide a significant increase in sensitivity for detection of anti-HCV. STUDY DESIGN AND METHODS: Twenty hemodialysis patients who were seroconverting to anti-HCV were included in this study. They were followed up for a mean period (+/− SD) of 10.5 +/− 3.3 months, in which 13 to 46 serum samples per case were collected. Each sample was tested for anti-HCV by second- and third-generation enzyme immunoassay (EIA-2 and EIA-3) and recombinant immunoblot assay (RIBA-3). E2 antibodies were tested by a prototype EIA in which E2 was expressed as a recombinant antigen in Chinese hamster ovary cells. RESULTS: Alanine aminotransferase elevation was observed in 18 of 20 cases. Reactivity against c100, c33c, c22, NS5, and E2 was detected in 15 (75%), 19 (95%), 15 (75%), 2 (10%), and 17 (85%) patients, respectively; c33c was the most immunogenic antigen, followed in descending order by E2, c22, c100, and NS5. E2 antibody reactivity resolved the two RIBA-3- indeterminate cases. However, there was no case in which E2 reactivity preceded all other HCV antigens. Anti-E2 was found to react in all patients of genotypes 1a, 1b, and 3a but in only 2 of 4 patients of genotype 4a. CONCLUSION: In this group of seroconverting individuals, E2 antigen was shown to be highly immunoreactive and did resolve some RIBA-3-indeterminate samples as being positive, on the basis of reactivity to multiple antigens, but it did not improve early detection of seroconversion.     

Application of a new enzyme-linked immunosorbent assay for detection of total hepatitis C virus core antigen in blood donors

Recent studies have shown that total hepatitis C virus (HCV) core antigen, both free and antibody bound, is an accurate indirect marker of viral replication that can be used in clinical practice. The aim of the present study was to evaluate the performance of a new total HCV core antigen enzyme-linked immunosorbent assay (ELISA) for detection and quantification of total core antigen in blood donors, testing positive for anti-HCV antibodies and for prospective low-risk population screening. A population comprising 257 samples, from blood donors detected reactive for anti-HCV antibodies [137 recombinant immunoblot assay (RIBA) positive and 120 RIBA indeterminate], were tested by using a new total HCV core antigen ELISA. HCV-RNA was quantified by using quantitative polymerase chain reaction (PCR) assays in all RIBA-positive samples and RIBA-indeterminate samples that were positive for the total core antigen. Specificity of the assay was studied in 1070 healthy blood donors negative for anti-HCV antibodies. Compared with quantitative PCR assays, the total HCV core antigen assay showed 97.37% sensitivity. The three HCV-RNA-positive samples, which tested negative for the total core antigen, had a low viral load (< 1.4 x 10(4) IU mL(-1)). All samples with more than 1.4 x 10(4) IU mL(-1) of viral RNA were positive for total core antigen, independent of the HCV genotype. Concentration of total core antigen correlated significantly with those of HCV-RNA (r = 0.614, P < 0.0001). Overall specificity in freshly collected blood donor specimens was 99.63%. Our data indicate that the total HCV core antigen ELISA has a sensitivity close to PCR assays in diagnosing HCV infection in blood donors with anti-HCV antibodies and shows an excellent specificity in volunteer donors. This assay, in combination with anti-HCV antibodies screening tests, could be an alternative to molecular assays for HCV infection screening in blood donors.     

Low-positive anti-hepatitis C virus enzyme immunoassay results: an important predictor of low likelihood of hepatitis C infection

BACKGROUND: Tests for hepatitis C antibodies (anti-HCV enzyme immunoassays) are usually described as positive or negative. Several studies, mainly in blood donors, have found that specimens with low signal/cutoff (S/C) ratios are commonly negative when tested with a recombinant immunoblot assay (RIBA) or for HCV RNA. METHODS: We retrospectively reviewed 17 418 consecutive anti-HCV results from a screening program for high-risk veterans; 2986 (17.1%) samples were anti-HCV-positive, and 490 (16.4%) had S/C ratios 相似文献   

Reliability of the third-generation recombinant immunoblot assay for hepatitis C virus

BACKGROUND: In a confirmatory laboratory, the second-generation recombinant immunoblot assay (RIBA-2) was replaced by the third- generation RIBA (RIBA-3) in March 1993. The aim of this validation study was to compare the sensitivity and specificity of RIBA-2 and RIBA- 3 in a routine setting, by using a validated hepatitis C virus (HCV) RNA polymerase chain reaction to establish plasma viremia. STUDY DESIGN AND METHODS: RIBA-2 testing was performed (March 1991-March 1993) in 593 HCV RNA-positive and 1498 HCV RNA-negative subjects. RIBA-3 testing was performed (March 1993-May 1994) in 220 HCV RNA-positive and 530 HCV RNA-negative subjects. All samples reacted for anti-HCV in enzyme- linked immunosorbent assay. RESULTS: In HCV RNA-positive individuals, the sensitivity of RIBA-3 was significantly higher than that of RIBA-2 (99.5% vs. 93.3%, p = 0.0005). This was not caused by inclusion of the NS5 antigen, but by a higher sensitivity of the antigens c33 and c100 (RIBA-2: 94.3% and 62.6%; RIBA-3: 99.5% and 88.6%). Replacement of the c22 and c100 recombinant proteins by synthetic peptides significantly reduced nonspecific reactivity against these antigens (p < 0.0001). Unfortunately, increased nonspecific reactivity against the modified c33 antigen and the new NS5 antigen canceled out this effect. Two-band reactivity occurred more often in nonviremic persons than in viremic persons (32.7% vs. 8.2%, p < 0.0001). Risk factors for HCV infection were less frequently observed in 11 blood donors with two-band reactivity than in 6 blood donors with other positive RIBA-3 patterns (18% vs. 83%, p = 0.03). CONCLUSION: The higher sensitivity of RIBA-3 significantly reduced the number of indeterminate test results in HCV RNA-positive persons. Confirmatory laboratories must be aware of the frequent occurrence of nonspecific, isolated reactivity and even nonspecific, two-band reactivity in anti-HCV enzyme-linked immunosorbent assay-reactive blood donors.