HCV感染后NS5区抗体的动态观察与检测意义

本文报道用ＨＣＶＮＳ５区两段抗原性较好的合成肽研制的ＥＬＩＳＡ试剂盒及ＵＢＩＨＣＶＮＳ５区抗体检测ＥＬＩＳＡ试剂盒观察１４例ＨＣＶ感染者ＮＳ５区抗体的动态变化，证实ＮＳ５区抗体如同ＮＳ４区抗体一样比Ｃ及ＮＳ３区抗体出现晚。ＮＳ５区抗体总体检出率近似于ＮＳ４区抗体；１．５５％的血清为单独ＮＳ５区抗体阳性；存在ＮＳ５区抗体与其他区抗体滴度有互补作用的标本等提示ＮＳ５区抗体仍有一定的诊断价值。采用ＵＢＩＮＳ５区抗体检测试剂盒发现，９５．６５％（２２／２３）ＵＢＩ试剂盒诊断为ＮＳ５区抗体阳性的标本中含ＨＣＶＲＮＡ，６／１４ＨＣｖ感染者用ＵＢＩ试剂盒检出的抗体出现在ＡＬＴ再次异常升高或剧烈升高（高于参考值的３倍以上）前后，４／１４的感染者抗体出现于ＡＬＴ首次升高前后（其余４／１４的感染者未检出抗ＮＳ５抗体），因此ＵＢＩ抗ＨＣＶＮＳ５抗体诊断试剂盒检测的抗体似与疾病的活动有关。     

初次及再次感染HCV后不同功能区抗体的研究

１４例初次ＨＣＶ感染者及１１例再次ＨＣＶ感染者系列血清系在无法检测ＨＣＶ时留存的２８９位手术受血者系列血清中筛选获得。系列血清包括受血前、受血后不同时期收集的血清。回顾性地研究其不同功能区抗体出现的动态变化，抗Ｃ与抗ＮＳ３抗体有早期诊断价值。抗ＮＳ３、抗Ｃ、抗ＮＳ５及抗ＮＳ４抗体在ＨＣＶ感染后系列血清中检出率分别为８４．７６％、７９．２７％、７２．５４％和６８．３９％。感染过程中各区抗体可以全部出现、部分出现或单独出现抗Ｃ、抗ＮＳ３及抗ＮＳ５抗体，未发现单独含抗Ｅ、抗ＮＳ１及抗ＮＳ４区抗体的血清。抗Ｅ、抗ＮＳ１及抗ＮＳ４抗体消失较早。研究表明：以ＨＣＶＣ区、ＮＳ３区及ＮＳ５区编码的优势抗原包被的ＥＬＩＳＡ抗体诊断试剂盒将提高ＨＣＶ的诊断。     

庚型肝炎病毒C型（GBV—C）多肽的抗原性研究及应用

应用Ｇｏｌｄｋｅｙ软件分析ＧＢＶ－Ｃ的氨基酸序列，采用标准的Ｍｅｒｉｆｉｅｌｄ固相法和多中心同步多肽合成技术研究ＧＢＶ－Ｃ多肽抗原，应用抗原反应性好的ＮＳ３区Ｐ１和Ｐ１１多肽以及ＮＳ５区的Ｐ２２多肽研制抗ＧＢＶ－Ｃ检测试剂。９９例ＧＢＶ－ＣＲＮＡ阳性血清的抗ＧＢＶ－Ｃ阳性率为８１．８２％（８１／９９），１０００例单采浆献血员血样的抗ＧＢＶ－Ｃ阳性率为５．１０％（５１／１０００），抗ＧＢＶ－Ｃ阳性血样中的ＧＢＶ－ＣＲＮＡ阳性率为７０．５９％（３６／５１）。结果表明，Ｐ１，Ｐ１１和Ｐ２２可以用于抗ＧＢＶ－Ｃ检测试剂的开发。应尽快对献血员，尤其是献浆员进行抗ＧＢＶ－Ｃ的筛选。     

检测抗丙型肝炎病毒总抗体的双抗原夹心法的建立

以丙型肝炎病毒（ＨＣＶ）结构区和非结构区基因工程表达抗原及人工合成多肽包被酶标板，用辣根过氧化物酶标记抗原，建立了检测血清中抗－ＨＣＶ总抗体的双抗原夹心法。与普遍采用的检测抗－ＨＣＶＩｇＧ的间接酶联免疫吸附试验（ＥＬＩＳＡ）比较，其灵敏度（１∶１２８）稍高于间接法（１∶６４），特异性相同，均为１００％。该法可同时检测抗－ＨＣＶ各类抗体，使检出率提高１０％。     

输血后丙型肝炎病毒非结构区抗体和丙氨酸转氨酶的动态分析

利用重组的丙型肝炎病毒非结构区（ＨＣＶＮＳ５）抗原建立了酶免疫试验（ＥＩＡ），对２５例输血后丙型肝炎进行了不同区抗体及丙氨酸转氨酶（ＡＬＴ）的动态研究，同时对１５６例慢性丙型肝炎患者血清进行ＨＣＶＲＮＡ和抗－ＮＳ５平行检测，两者符合率为６４．１％。抗－ＮＳ５抗体首次检出时间为３０～５７５天（１８２．９±１６８．５），晚于ＡＬＴ异常和其他区抗体的出现时间。在感染后１，３，６，１２和２４个月后抗－ＮＳ５的阳性率分别为２８％，４０％，５２％，６８％和７６％。抗－ＮＳ５的动态变化类型为四种：一过性阳性、间歇性阳性、持续性阳性和２年内持续阴性     

母婴血清中丙型炎病毒抗体的检测

本文应用酶联免疫吸附试验（ＥＬＩＳＡ）检测了１４７对母婴血清丙型肝炎病毒抗体（抗－ＨＣＶ），产妇抗－ＨＣＶ阳性率为４．７６％，新生儿抗－ＨＣＶ阳性率为２．０３％，线婴抗－ＨＣＶ均阳性者２对，阳性率为１．３６％。     

五种丙型肝炎病毒(HCV)基因重组抗原斑点酶免疫反应检测血清抗-HCV

五种丙型肝炎病毒（ＨＣＶ）基因重组抗原斑点酶免疫反应检测血清抗－ＨＣＶ谭德明刘双虎胡国龄刘安国刘国珍应用五种丙型肝炎病毒（ＨＣＶ）基因重组抗原，包括ＨＣＶ核心蛋白（Ｃｏｒｅ），包膜蛋白１（Ｅ１）、包膜蛋白２（Ｅ２／ＮＳ１）、ＮＳ３和ＮＳ５抗原〔１，２...     

丙型肝炎病毒NS3蛋白单克隆杂交瘤细胞株的建立及初步鉴定

应用国产基因工程表达的丙型肝炎病毒（ＨＣＶ）ＮＳ３区抗原免疫小鼠，然后取其脾细胞与小鼠骨髓瘤细胞系ＳＰ２／０融合，筛选出４株稳定分泌抗ＨＣＶＮＳ３区蛋白单克隆抗体（ＭｃＡｂ）的杂交瘤细胞株，分别命名为２Ｂ６，２Ｆ３，３Ｄ８，３Ｄ９，经初步研究表明这４株单抗与ＮＳ３抗原具有良好的反应性，与ＨＣＶ核心区多肽及乙型肝炎病毒表面抗原和ｅ抗原均无反应。抑制实验表明这４株抗体分别针对ＮＳ３抗原分子上的２个不同的抗原决定簇。     

肝细胞肝癌中丙型肝炎病毒C区、E区、NS3区、NS4区抗原的表达

本文报道研究丙型肝炎病毒抗原在肝细胞肝癌组织内的定位分布情况。以丙型肝炎病毒（ＨＣＶ）的Ｃ、Ｅ、ＮＳ３、ＮＳ４区四种单克隆抗体用免疫组化方法检测了１３９例肝细胞肝癌（ＨＣＣ）的肝脏标本，结果总的阳性率为１５．１％。２１例阳性标本中，Ｃ区单抗检测阳性占８０．９％（１７／２１），Ｅ区占３３．３％（７／２１），ＮＳ３、ＮＳ４区均占５７．１％（１２／２１），表明应用多区段单抗有助于提高ＨＣＶ抗原的检出率。阳性物质主要存在于胞浆中，呈细、粗颗粒及块状，３例出现膜及膜下型，１例核内有阳性反应。ＨＣＶ感染与ＨＣＣ的发生发展有一定的关系。     

用重组抗原检测鼻咽癌病人血清IgA／gp125抗体

用重组抗原检测鼻咽癌病人血清ＩｇＡ／ｇｐ１２５抗体薛绍礼１皮国华２谷淑燕２李平１抗Ｅｐｓｔｅｉｎ－Ｂａｒ病毒（ＥＢＶ）壳抗原（ＶＣＡ）ＩｇＡ抗体，是鼻咽癌（ＮＰＣ）早期诊断的一个重要血清学指标。曾先后用免疫荧光法（ＩＦ）和免疫酶法（ＩＥ）进行检测［１...     

输血后丙型肝炎病毒非结构区抗体和丙氨酸转氨酶…

In order to study the character and dynamic changes of HCV NS5 antigen in post-transfusion hepatitis C, EIA was established with fusion protein, and different groups of population were investigated. The results showed the positive rate of whole blood donor, normal people, chronic hepatitis C patients and post transfusion hepatitis C patients is 0.0%, 1.66%, 50.7% and 70.5%, respectively. Serial blood samples of 25 cases of acute or chronic post-transfusion hepatitis C were detected, and the results indicated that the antibody of NS5 appeared relatively late, and the serum conversion time was 182.9 +/- 168.5 days. The anti-NS5 positive rate in 1, 3, 6, 12 and 24 months after HCV infection was 28%, 40%, 52%, 68% and 76%, respectively. The positive rate of anti-NS5 in sera with HCV RNA was 61.9%. Analysis on the appearance of antibody to the different antibody region of the gene, ALT and HCV RNA showed the dynamic changes of anti-NS5 were corresponding to the serum ALT in some cases, indicating antibody of NS5 appear later and may reflect the disease activity to some extent. The dynamic changes of anti-NS5 are of four kinds: transient positive, intermittent positive, persistent positive and persistent negative in during the two years period.     

丙型肝炎病毒E1抗体检测及临床应用

目的：研究丙型肝炎患者血清中HCV E1抗体，确定HCV E1抗原在抗体检测中的应用价值。方法：应用酶联免疫吸附测定（ELISA）方法检测80份卫生部第3代HCV血清参比品，821例职业献血员血清和720例临床肝炎患者血清中E1抗体。结果：用E1抗原单包板检查80份卫生部血清参比品的阳性符合率为70％、阴性符合率为100％；从821例职业献血员血清样品中检出E1抗体阳性率为1．9％；从720例临床肝炎患者血清中检出E1抗体阳性率为68％。大部分E1抗体阳性血清，同时也能和HCV的Core、NS3抗原及NS5A抗原呈阳性反应，但有个别血清只对E1抗原呈阳性反应。用市购HCV抗ELISA检测试剂盒检测为阴性的218例肝炎科门诊患者、813例献血员和848例一般人群血清，用E1抗原单包板复检，检出的阴性率分别为1．4％、1．1％和0．9％。在3例患者血清学转变的追踪研究中，HCV E1抗体都同现最早。结论：用E1工程蛋白检测E1抗体具有较高的灵敏度和特异性。E1抗体在HCV感染患者中普遍存在而且早出现，在临床诊断上是有意义的。     

单纯GBV-C/HGV感染人体血清学和病理学追踪研究

目的 从临床和病理学方面探讨庚型肝炎病毒（GBV－C／HGV）的致病性。方法 收集24例单纯血清GBV－C／HGV RNA阳性人体的穿刺活检肝组织及血清标本,其中8例作间隔2年以上的二次肝穿,进行血清和肝组织GBV－C／HGV RNA、血清抗E2抗体及ALT水平、肝组织NS3和NS5抗原检测,并作肝组织光、电镜观察。结果 24例血清GBV－C／HGV RNA阳性者首次肝穿前3d内平均ALT水平为60．17IU／ml(42-87IU/ml),抗E2抗体阳性率4．17％,首次肝组织GBV－C／HGV RNA阳性率为75．00％,NS3和（或SN5抗原阳性率为54．17％。GBV－C／HGV RNA及NS3和NS5抗原主要于肝细胞质内检出,阳性细胞呈散在分布,少数浸润的单个核细胞内有病毒RNA检出。肝细胞呈极轻度急、慢性炎症病变者占79．17％。与2年前比较,2年后24例观测对象血清GBV－C／HGV RNA自然转阴率66．67％（P＜0．001）,血清ALT复常率75．00％（P＜0．001）,E2抗体阳性率为41．67％（P＜0．001）,8例二次穿刺肝组织除2例有灶状肝细胞水样变性外,余均复常。结论 庚型肝炎病毒可引起极轻度自限性肝炎,提示其致肝损伤作用微弱且具有自限性;血清E2抗体是GBV－C／HGV感染恢复性标志,是否存在其他恢复性血清标志物尚待研究。     

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     

A novel antigen detection immunoassay for field diagnosis of hepatitis C virus infection

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 (approximately 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.     

Elevata prevalenza ma bassa incidenza della infezione da HGV in pazienti con epatite cronica C

Objectives To determine 1. The prevalence and incidence of HGV infection in patients with chronic hepatitis C and 2. Its influence on the clinical outcome of chronic hepatitis C. Patients and methods Sixty-five patients with non-parenteral chronic hepatitis C virus infection were investigated for HGV infection using the polymerase chain reaction for HGV-RNA and by detecting serum antibodies against the E2 protein of HGV (anti-E2 antibodies). Results HGV-RNA in serum was found in 12 patients (18.4%) and anti-E2 antibodies in 4 (6.1%). No difference in age, sex, liver histology, basal ALT or ?GT was found between HGV positive and negative cases. Thirty-four patients (6 with HGV-RNA) were followed-up for 4 years; 4 of the 6 lost HGV-RNA, one of whom seroconverted to anti-E2. None of the 28 HGV-RNA negative cases presented HGV infection during the follow-up period. The presence of HGV infection did not influence either basal HCV viremia or the response of HCV to IFN therapy. Conclusions The study demonstrated that HGV had an intense circulation through non-classic parenteral routes, but its impact on HCV replication and liver disease is negligible.     

原位杂交法检测非甲～庚型肝炎患者肝组织中TT病毒DNA

目的 证实在非甲－庚型病毒性肝炎患者肝组织中TT病毒（transfusion-transmitted virus,TTV)的存在。方法 采用地高辛素标记TTV DNA探针以原位杂交技术对51例血清学病毒标记非甲－戊型、免疫组化检测肝组织中HBsAg、HCV NS3Ag及HGV N55Aag阴性的病毒性肝炎患者石蜡包埋肝组织进行了检测。结果 各型病毒性肝炎肝组织中TTV基因的总检出率为27．5％,其中急性轻型肝炎的检出率为30．8％（4 ／13）,急性重型肝炎为1／8,亚急性重型肝炎为3／7,慢性肝炎为2／6,活动性肝硬化为2／9,慢性重肝肝炎为1／4,原发性肝癌为1／4。TTV DNA表达于肝细胞核或胞质内,以核型多见。在急性肝炎,TTV阳性细胞弥漫分布于肝小叶内,慢性肝炎于汇管区附近较为密集,而在肝硬化病例,阳性细胞在假小叶内多呈片族状不规则分布。结论 在不明原因病毒性肝炎患者血清及肝组织中TTV DNA的检出表明TTV为一种新型的肝炎病毒,TTV为一种嗜肝性病毒。     

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.     

Correlation of interferon treatment response with GBV-C/HGV genomic RNA and anti-envelope 2 protein antibody

The clinical significance of GB virus C/hepatitis G virus (GBV-C/HGV) co-infection was studied retrospectively in 100 consecutive patients with hepatitis C virus (HCV) infection. All 100 patients had been treated with interferon-alpha (IFN-alpha). Co-infection with GBV-C/HGV and HCV was detected in 10 of the 100 patients (10%) and anti-envelope 2 region (anti-E2) antibody was detected in 25 patients. None of the patients with GBV-C/HGV RNA had anti-E2 antibody. Co-infected patients were younger (P < .005) and their serum transaminase levels were lower than HCV-only infected patients (P< .01). In 7 of the 10 co-infected patients, HCV RNA was eradicated from serum after IFN-alpha treatment and normal alanine transaminase (ALT) levels continued in 6 of these 7 patients. In one patient who was negative for HCV RNA but positive for GBV-C/HGV RNA, the ALT level relapsed transiently. The rate of clearance of HCV and normalization of the ALT level was significantly higher in co-infected patients than in HCV-only infected patients (P < .05). GBV-C/HGV RNA disappeared from 6 of the 10 co-infected patients (60%) upon cessation of IFN-alpha treatment. However, continuous clearance of GBV-C/HGV was observed in only two patients and anti-E2 antibody could not be detected in the serum of these patients. These results indicate that co-infected patients tend to be younger and more sensitive to IFN-alpha treatment. However, long-term clearance of GBV-C/HGV after IFN-alpha treatment may be difficult. Moreover, anti-E2 antibody may act to neutralize GBV-C/ HGV.     

Anti-HCV RIBA/LiaTek reactivity and HCV genotype in EIA-negative patients with viremia.

Individuals infected with hepatitis C virus (HCV) usually produce anti-HCV antibodies detectable by enzyme immunoassay (EIA); however, in certain viremic cases this antibody does not appear. To investigate whether anti-HCV in these cases is detectable by Western blot (WB), 38 HCV RNA positive/anti-HCV EIA-negative sera were tested by RIBA 3.0 or LiaTek III. The HCV genotypes (INNO-LiPA) were analyzed to determine whether the variance in these genotypes can be the reason for the late, weak antibody production or its absence. As the control group, 282 EIA-positive/HCV RNA-positive patients were examined. A single band reactivity of various intensities by RIBA or LiaTek was observed in 16/38 EIA negative sera. Positive results with NS3 were detected in 4 sera and weak positive (+/-) with core, NS3, and NS5 in 5, 6, and 1 sera, respectively. In 3 cases with anti-NS3, the seroreversion was observed in follow-up. The distribution of genotypes in anti-HCV-negative versus anti-HCV-positive groups was: 1b alone, 50.0% vs. 78.0%; 3a alone, 13.2% vs. 15.6%; and mixed (1b+3a), 36.8% vs. 5.0%, respectively. The follow-up studies showed that viremia was lost spontaneously in 12/35 patients. In some patients infected with two genotypes, the spontaneous loss of the 3a genotype was observed. The study showed that WB tests are useful for serological confirmation of HCV infection in some EIA negative/HCV RNA-positive patients but, because seroreversion may occur, sequential sera samples should be tested. No unusual HCV genotype was detected in anti-HCV-negative/HCV RNA-positive cases, but the frequency of mixed infection with the 1b+3a genotypes in this group was found to be higher than that in anti-HCV-positive hepatitis patients.