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。     

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。     

丙型肝炎病毒抗体化学发光检测方法的建立

丙型肝炎是由丙型肝炎病毒(hepatits C virus,HCV)引起肠道外传播的一类传染病,输血及血液制品引起传播是本病的主要途径。目前,国内抗HCV检测主要是ELISA和进口的全自动化学发光法。进口的全自动化学发光检测,由于配套的全自动分析仪和试剂价格昂贵,限制了其在临床上的应用。本研究研制了一种更为便宜、更易操作的HCV抗体化学发光试剂盒。     

酶联免疫吸附试验在丙型肝炎抗体检测中质量控制探讨

寻找适合酶联免疫吸附试验对丙型肝炎抗体检测的室内质量控制(internal quality controlling,IQC)的方法。采用Levey-Jennings质控图法、即刻法、加强即刻法(CV30%)对相同的20例数据进行处理。再用双质控法处理更换试剂批号和血清质控。和传统的质控方法比较,Levey-Jennings质控图法和即刻法对数据监控上均存在假失控和假在控。而加强即刻法显示良好效果。配合双质控法处理更换试剂批号和血清质控也很优秀。依据提出的质控方法进行室内质控,其当年室间质评结果全部合格。课题组的结论是控制变异系数的加强即刻法配合L-J质控图法更适用于酶联免疫吸附试验的IQC。     

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％。     

庚型肝炎病毒IgM抗体检测方法的建立及其临床意义

目的建立一种早期、快速诊断庚型肝炎的血清学检测方法。方法以辣根过氧化物酶标记庚型肝炎病毒（ＨＧＶ）多肽ＮＳ３,ＮＳ５区段抗原,建立了捕获酶联免疫吸附试验（ＥＬＩＳＡ）,用于检测血清中ＨＧＶＩｇＭ抗体。结果本法不受特异性ＩｇＧ的竞争和类风湿因子的干扰;与其它致肝炎的病毒（ＨＡＶ、ＨＢＶ、ＨＣＶ、ＨＥＶ、ＣＭＶ、ＥＢＶ）无交叉反应。检测４６例非甲、乙、丙、戊型肝炎患者血清,抗－ＨＧＶＩｇＭ阳性１４例,阳性率３０．４３％,其中,６例同时为ＨＧＶＲＮＡ阳性,阳性符合率为４２．８６％（６／１４）,检测１２例庚型肝炎病人双份血清,其中,急性期血ＨＧＶＩｇＭ抗体均为阳性。结论该法检测ＨＧＶＩｇＭ抗体特异性强,敏感性高,且简便快速,适用于临床对庚型肝炎新近感染的早期诊断,有推广应用价值。     

蛋白芯片检测丙型肝炎病毒分片段抗体的临床意义

丙型肝炎是一种严重威胁人类健康的世界性血源性传染病，加强血源及血液制品的监控是有效防止感染丙肝病毒的重要手段。目前检测丙型肝炎以检测血清中抗HCV抗体的ELISA法较为普遍，但仍有不足之处。     

非平衡法在乙型肝炎e抗体和核心抗体检测中的效果评价

目的 探讨酶联免疫吸附试验非平衡法对乙型肝炎e抗体和核心抗体检测结果的影响.方法 收集电化学发光免疫分析法（ECLIA）检测抗-HBe阳性标本44例和抗-HBc阳性标本57例,再用酶联免疫吸附试验法（ELISA）对阳性标本进行检测,样本加入反应孔中分别于0min、30min后加入酶标志物,余下步骤均严格按照说明书操作.结果 对ECLIA检测抗-HBe阳性标本44例、抗-HBc阳性标本57例进行ELISA复检后,平衡法抗-HBe的漏检率为52.3％,抗-HBc漏检率为40.4％;非平衡法抗-HBe的漏检率为34.1％,抗-HBc漏检率为24.6％.结论 竞争法检测抗-HBe和抗-HBc时应适当延迟加酶的时间,以降低漏检率.     

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…     

HCV antibody test methods and patterns of antibody response]

The sensitivity and specificity of the second generation ELISA (2nd ELISA) and RIBA (2nd RIBA) using c 22 and c 200 or c 33 c recombinant antigens encoded in the core and NS 3 of HCV genome have been evaluated preclinically by Ortho and Chiron (USA). Sensitivity of the assay was evaluated by determining the reactivity of 2nd ELISA and RIBA in the following panels: NANBH (N = 35) and high risk groups composed of: hemophiliacs (N = 50), IVDA (N = 50) and renal dialysis cases (N = 279). In the reactive specimens, antibody response to both c 33 c and c 22-3 was present in 93-100% whereas antibody response to 5-1-1 and c 100-3 was seen in 46-84% of these same specimens. Specificity, in turn, was evaluated using 2000 volunteer donors of 29 (1.45%) 2nd ELISA positives, 16/29 (55.2%) were confirmed by 2nd RIBA and 8/29 (27.6%) were 1 band positive (indeterminate), whereas of 27 (1.35%) 1st ELISA positive, 9/27 (33.3%) were confirmed and 4/27 (14.8%) were 1 band positive by 1st RIBA (c 100-3, 5-1-1). The relevance of various band patterns and correlation between PCR positivity with 2nd RIBA reactivity are discussed. The consistency and sensitivity of the 2nd RIBA evaluated using a densitometer are also reported.     

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     

Analysis of HCV-immunoglobulin isotype complexes provide new insights into antibody response to HCV

Hepatitis C virus (HCV) is known for its ability to establish persistent infection and cause chronic hepatitis in most infected individuals. The antibody response to HCV in HCV-circulating immune complexes (CIC) is unknown. In the present study, we have characterized distinct changes in patterns of HCV-immunoglobulin (Ig) constituents with disease category, viral mutation and clinical markers. The number of samples positive for single HCV-Ig, HCV-IgG and HCV-IgA, HCV-IgM and HCV-IgA, HCV-IgM and HCV-IgG, HCV-IgM, HCV-IgG and HCV-IgA in 47 samples tested were 8 (17%), 1 (2.1%), 9 (19.1%), 4 (8.5%) and 17 (36.2%), respectively. The occurrence of HCV-IgM and HCV-IgA in combination of two isotypes of HCV-Ig became predominant. These results show that defective IgG in HCV-CIC may contribute to long-term viremia. Further analysis indicated that the frequency of HCV RNA/IgA-CIC in the abnormal aspartic aminotransferase (AST) group was significantly higher than that of the normal AST group, and HCV RNA/IgA-CIC frequency in the abnormal alanine aminotransferase (ALT) group was slightly higher than that in the normal ALT group. IgA complexes may reflect the damage degree of liver function during the course of HCV infection. We also found that there were more mutations in supernatant than in other constituents from single-strand conformation polymorphism (SSCP) analysis. Our results suggest that Ig-complexed virions and free virions may have different biological consequences, with the latter being elusive to immunological elimination. The findings in this study may provide some new insights into antibody response to HCV.     

HCV NS3蛋白单克隆抗体的制备及其识别区域的分析

目的 制备针对丙型肝炎病毒（HCV）非结构区NS3全长蛋白的单克隆抗体（MAb）,并分析获得的单抗识别表位所在区域,为建立以NS3蛋白为靶位的抗HCV研究提供抗体工具。方法 用原核表达的HCV非结构区NS3全长蛋白作为免疫原,采用小鼠腹股沟皮下NC膜包埋法免疫小鼠,按常规杂交瘤细胞的制备方法,经细胞融合、克隆化制备抗NS3蛋白的MAb。用间接免疫荧光法和Westem blot鉴定其特异性。分别构建NS3丝氨酸蛋白酶（NS3蛋白的N末端1／3）编码基因的真核表达质粒pcDNA3．1（-）-ns3p、NS3解旋酶（NS3蛋白的C末端2/3）编码基因的真核表达质粒pcDNA3．1（-）-ns3A,将其瞬时转染COS-7细胞后,以获得的单克隆抗体作为一抗,通过免疫荧光分析获得单抗识别表位所在的区域。结果 获得了2株抗NS3蛋白的单克隆抗体,这2株MAbs均特异识别NS3蛋白,并确定了它们的结合区域。结论 获得了针对NS3蛋白的单克隆抗体,并对其单抗识别表位所在的区域进行了分析,为下一步进行以NS3蛋白为靶位的抗HCV研究奠定了良好的基础。     

Use of single radial haemolysis for assessing antibody response to influenza virus vaccines in animals

The value of the single radial haemolysis (SRH) test as a possible replacement for the haemagglutination-inhibition (HAI) test for the estimation of antibodies against influenza was assessed in three animal models. The serum antibody response was measured by both assay systems; correlation of the two tests was assessed using regression analysis. The study showed that when the response to a single immunisation was determined, the ferret model gave satisfactory correlation of SRH and HAI, whilst in the mouse and hamster models poor correlation was observed. Correlation was only improved in the mouse model when an immunisation schedule that mimicked the human situation of a background exposure to different strains of influenza virus was used. Since influenza vaccine efficacy is usually assessed in animals using a single immunisation we suggest that the SRH is not acceptable for use in either hamsters or mice, but is acceptable where the ferret model is involved.     

鼠抗HCV单克隆抗体研制及初步应用探讨

交联HCV 合成多肽抗原,用鼠一鼠杂交瘤技术获得8株稳定分泌抗HCV 的特异性单抗,分别命名为YLCV 系1—8。经初步研究结果表示,抗HCV 抗体阳性血清可抑制酶标YLCV-1和YLCY-5单抗与抗原的反应。抗原测定结果6份血清中有一份呈强阳性反应,由此提示单抗在HCV 抗体抗原检测系统中应用的可能性,同时为HCV 抗原成份的分析提供了必要的条件.     

Functional convergence of a germline-encoded neutralizing antibody response in rhesus macaques immunized with HCV envelope glycoproteins

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Detection of IgE antibody specific to a hepatitis C virus-derived peptide being recognized by cellular immunity in patients with HCV infection

The determination of immunogenic peptides of hepatitis C virus (HCV) is pivotal for vaccine development. We previously reported that the majority of patients infected with HCV have significant levels of IgG specific to an HCV-derived peptide at positions 35-44 of core protein (C35-44), a major epitope recognized by cellular immunity. This study addresses whether or not the other subclasses of immunoglobulins to this peptide exist. As a result, IgE, but not IgM or IgA, specific to this peptide is consistently detectable in the majority of patients with HCV infection, regardless of the different HLA types and disease conditions. These results provide additional information on this immunogenic peptide with new insights that contribute to a better understanding of host responses to HCV.     

抗人血管生长素单链抗体可变区基因的定向克隆及序列分析

血管生长素（ａｎｇｉｏｇｅｎｉｎ,ＡＮＧ）是一种存在于正常人血浆及实体肿瘤组织中的蛋白质,它具有很强的促血管生长作用。大量研究表明：ＡＮＧ与肿瘤的快速生长和转移有密切的关系＾〔１〕。ＡＮＧ单抗可以通过中和ＡＮＧ或阻断ＡＮＧ与血管内皮细胞受体的结合而实现其抑瘤作用＾〔１〕,但传统的杂交瘤技术制约了ＡＮＧ单抗的制备,而基因工程抗体技术具有克隆筛选简捷、抗体制备方便及易进行抗体基因修饰操作等优点。本文通过抗人ＡＮＧ单链抗体可变区基因的定向克隆和序列分析的研究,将为临床提供一种经济有效的新型抗体打下基础。     

The serology of hepatitis C virus (HCV) infection: antibody crossreaction in the hypervariable region 1

Summary We determined the NS1/E2 N-terminal sequence including the hypervariable region 1 (HVR1) from five individuals chronically infected with HCV: two from the Czech Republic and three from Germany. From each sequence, six 12-mer overlapping peptides were synthesized and used in a peptide scan to evaluate seroreactivity of each of those patients, as well as three anti-HCV positive blood donors to the different isolates. We could show the general presence of antibodies to multiple HVR1 specific sequences reflecting the existence of multiple variants in infected persons. Finally, we observed the persistance of HCV infections in all individuals despite an active humoral response directed against the virus.