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1.
戎广亚  孙杰 《病毒学报》1998,14(2):178-182
用ORF2、ORF3合成肽抗原(1-10号)及基因工程重组的ORF2抗原(1和2号)分别建立了酶联免疫方法(EIA),检测60份戊型肝炎病人血清中HEVIgG及IgM10个合成肽抗原及2人重组抗原,均和HEV阳性血清发生特异反应,但阳性率和反应强度差别很大。以Rel(ORF2,402-660)检测的抗体阳性率最高,为96.7%;Sp6(ORF3,88-123)次之,为93.3%(56/60);以上  相似文献   

2.
人类免疫缺陷病毒1/2型抗体检测酶联免疫试剂盒的研制   总被引:2,自引:0,他引:2  
采用二聚体合成肽(HIV-1gp41、gp120、p24和HIV-2gp36)包被酶标板条制备成固相抗原,与鼠抗人IgG单克隆抗体酶标记物、底物TMB及阴阳性参考血清配套制备成HIV抗体EIA试剂盒,专供检测人血清或血浆HIV1/2抗体之用。以荷兰、韩国及万泰试剂作为对照,用该试剂盒对检定所的Panel标准及献血员15550例(其中HCV抗体阳性128例,HBsAg阳性46例)进行检测,四种试剂对检定所Panel标准的13份阳性血清均呈阳性反应,28份阴性血清均为阴性;献血员15550例,四种试剂对其中1份血清均呈阳性反应,经Westernblot试验证实为阴性,四种试剂的阴性检出率均为99.99%。连续制备三批试剂经中国药品生物制品检定所检定,所检项目全部合格;同时委托检定所进行临床考核,47份阳性血清全部呈阳性反应,150份阴性血清全部为阴性。说明该试剂盒具有很好的敏感性和特异性。  相似文献   

3.
用人工合成的丁型肝炎病毒抗原(HDV-Ag)肽建立了检测抗HDV-IgM抗体的ELISA方法,本法操作简便、快速,重复性好,特异性强,与抗HAV-IgM、抗Hk-IgM、抗HBs-IgM、抗HCV-IgI、抗CMV-IgM、抗RV-IgM、类风湿因子(RF)及抗核抗体(ANA)阳性血清均不起反应,且可被2-巯基乙醇阻断而不起反应。经初步临床应用,31例正常人血清抗HDV-IgM全部阴性,28例慢活肝患者检出率为32.1%(9/28),17例慢迁肝患者血清阳性率为11.8%(2/17)18例肝癌和肝硬化病人血清阳性率为22.2%(4/18)这三组病人与正常对照者相比较均有显著性差异(P<0.001)。此外,抗HDV-IgM阳性血清的ALT值均明显高于正常参考范围,提示在HDV感染过程中,患者肝细胞进一步受损。实验结果证明,抗HDV-IgM是诊断HDV感染的重要指标,对HDV感染早期诊断具有重要价值。  相似文献   

4.
应用ELISA和PCR法检测502例乙肝病人血清,401例HBsAg阳性血清中,有114例(28.4%)抗-HCV和HCVRNA双项阳性,25例(6.2%)HCVRNA单项阳性;21例(5.2%)抗-HCV单项阳性。将HBsAg乙肝病人分成HBVDNA,HBeAg阳性组和HBVDNA,HBeAg阴性组。前者抗-HCV阳性率为11.6%~20.5%,HCVRNA阳性率为16.2%~20.5%。后者抗-HCV阳性率为20.2%~55.6%,HCVRNA阳性率为23%~60.3%。结果说明长期携带HBV者和慢性乙肝病人均可重叠HCV感染。HBVDNA阳性组抗-HCV和HCVRNA阳性率明显高于HBVDNA阳性组  相似文献   

5.
将编码丙型肝炎病毒(HCV)E2蛋白417~750位氨基酸的DNA片段 克隆到真核表达载体pcDNA 3.1(-)中的CMV IE启动子下游,构建成HCV E2重组真核表达质粒 pcE2。ELISA法检测pcE2 DNA免疫兔血清中的E2抗体变化和维持规律,结果显示免疫20d已有 抗体产生,30d后开始进入高峰,40d时达到最高值,至第90d抗体水平保持平稳,抗体滴度 达到1∶1600左右。流式细胞计数仪(FACS)检测pcE2 DNA免疫鼠CD4+、CD8+T淋巴细胞变 化情况,与注射空载体pCDNA3.1(-)的阴性鼠相比,CD4+淋巴细胞水平略有上升,CD8+ 细胞水平有较大升高,增幅达35.46%。免疫组化检测结果显示注射pcE2的小鼠组织中有明显 的阳性着色,而注射pcDNA3.1(-)的对照组小鼠免疫组化结果为阴性。以上结果表明:pcE2 在实验动物内表达出的HCV E2蛋白可以引起免疫动物的体液免疫应答和细胞免疫应答,尤其 是MHC-1限制性杀伤性CD8+T淋巴细胞水平的提高对清除 病毒是十分有利的,因此HCV E2 DNA免疫有可能成为预防和治疗HCV感染的一条新途径。  相似文献   

6.
戊型肝炎病人血清抗—HEV IgG与IgM和HEV RNA的动态变化   总被引:13,自引:2,他引:11  
戎广亚  孙杰 《病毒学报》1998,14(3):268-271
利用酶联免疫试验(EIA)及逆转录-聚合酶链反应(RT-PCR),检测了210份急性非甲非乙非丙肝炎患者血清和40例戊型肝炎(戊肝)病人系列血清。在急性非甲非乙非丙肝炎血清中,抗-HEV IgG、抗-HEV IgM和HEV RNA阳性率分别为62.86%、45.23%和40.48%。在戊肝系列血清检测中,抗-HEV IgG阳性率发病1个月内为92.5%,发病2 ̄6个月100%,12个月94.7%,  相似文献   

7.
用抗单纯疱疹病毒(HSV)型共同性gC和gD单克隆抗体(McAb),包被Eppendorf管,捕捉HSV,同时加入3个引物:一个是HSV-1/HSV-型共同性上游引物,另两个分别是HSV-1和HSV-2型特异性下游引物。借此建立了能直接分型检测HSV的抗原捕获聚合酶链式反应(AC-PCR)。HSV-1的扩增产物为477bp,HSV-2的为399bp,两型病毒经AC-PCR扩增后产生分子量不同的DN  相似文献   

8.
新型HCV EIA诊断试剂盒的研制   总被引:3,自引:0,他引:3  
杨永平  曹经缓 《病毒学报》1994,10(2):118-127
丙型肝炎病毒(HCV)基因组结构区核壳蛋白(C)区抗原、膜蛋白E1和E2区抗原,以及非结构区NS3-NS5区抗原的区段,已经在原核细胞中获得有效的表达。同时,相应区段中的优势抗原表位肽也经化学合成法大规模地制备。HCV基因组上各区段抗原性的分析发现,由C区和NS3区分别编码的C抗原和C33c抗原是HCV基因组上两个优势抗原区段。其相应的抗体出现早(感染后6周可检出抗C33c抗体),阳转率高(约99%阳性检出率),特异性和重复性均优于其它区段抗原。以中国人HCV的C33c重组蛋白和分支状合成肽MAP-C-19为复合抗原,研制了适合我国抗HCV抗体检测的新型丙型肝炎病毒酶免疫测定(HCVELA)诊断试剂盒。它同当代美国Abbott/UBIHCVELA诊断试剂的符合率约98%,同加拿大YES公司HCVEIA诊断试剂的符合率约97.8%,阳性检出率提高了约2%,3次重复性达100%,表明其特异性、敏感性和重复性均达到了当代第二代JCVELA诊断试剂的水平。我国人群中抗HCV抗体的分布情况为:正常人群的检出率1%-2%;外科类住院病人检出率约28.8%;肝炎患者抗HCV阳性率为34.4%,慢活肝、肝硬化和重症肝炎患者  相似文献   

9.
采用固相法合成HIV-1和HIV-2两个多肽,建立了用混合多肽为包被抗原检测HIV-1和HIV-2感染的间接酶联免疫吸附法。检测46份抗HIV-1和HIV-2抗体阳性血清标本以及94份对照血清标本,与UBI试剂比较,其阳性符合率为97.8%,阴性符合率为100%,总符合率为99.3%。实验结果表明,此法可用于HIV-1和HIV-2感染的检测。  相似文献   

10.
单纯疱疹病毒(HSV)Ⅰ型及Ⅱ型之间有很多共同抗原,能引起血清学交叉反应,鉴别诊断比较困难。本实验利用重组DNA技术,将部分HSV-2DNA的PstI片段克隆到载体质粒PSK中,并筛选出两个重组质粒(P和P)只与HSV-2反应,与HSV-1不反应,这两个重组质粒中所含的HSV-2DNA片段大小分别是3.1和4.3kb,另外,还筛选了一个重组质粒(PHSV2-1,含5.8kbHSV-2DNA片段)与HSV-1和HSV-2均反应。将4.3kb的片段用光生物素标记后作为探针检测了159份人阴道拭子,其中23份样品呈阳性反应,其余均为阴性,从23份阳性样品中挑选12价涂片用间接荧光抗体法检测也都呈阳性反应,随机挑选的几份杂交反应阴性样品在间接荧光试验中也是阴性。本实验制备的HSV通用及HSV-2型特异性探针将比常规的血清学方法诊断和鉴别HSV-1和HSV-2感染更为可靠。  相似文献   

11.
Hepatitis E virus (HEV) infects both humans and animals, with an overall human mortality rate generally less than 1%, but as high as 20% among pregnant women. HEV strains fall into 4 major genotypes. Zoonotic genotypes 3 and 4 associate with sporadic human and animal HEV cases in many industrialized countries. To date, collective evidence implicates pigs as the main HEV reservoir, justifying the importance of monitoring HEV infection rates in pig herds to prevent human illness. Due to the lack of a robust in vitro cell culture system for viral propagation, no “gold standard” assay has yet been developed to detect HEV infection in domestic pigs. 1E4, a monoclonal antibody (mAb) specific for the C-terminal 268 amino acids of HEV genotype 4 ORF2 capsid protein (sORF2-C), was generated and conjugated to horseradish peroxidase (HRP) for use in a blocking ELISA (bELISA). Optimal sORF2-C coating antigen concentration (8 μg/ml), HRP-1E4 dilution (1:1000), and test pig serum dilution (1:20) were determined using a checkerboard titration test. A cut-off value of 16.9% was chosen to differentiate between positive vs. negative sera after mean percent inhibition (PI) testing of 230 negative pig sera. Compared with the indirect ELISA (iELISA), western blot, and a commercial ELISA kit for detecting anti-HEV antibodies in human sera, the bELISA showed no statistical differences and statistically high coincidence of 93.23%, 92%, and 95% with the other tests, respectively. A blocking ELISA (bELISA) for detecting anti-HEV antibodies in pig serum samples was developed with high sensitivity and high specificity comparable to that of the indirect ELISA. The bELISA results exhibited high agreement with iELISA, western blot, and a commercial ELISA kit designed to detect human anti-HEV antibodies. Therefore, bELISA should serve as an ideal method for large-scale serological investigation of anti-HEV antibodies in domestic pigs.  相似文献   

12.

Background

In recent years various studies showed, that hepatitis E virus (HEV) is a growing public health problem in many developed countries. Therefore, HEV infections might bear a transmission risk by blood transfusions. The clinical relevance still requires further investigations. The aim of this study was to provide an overview of acute HEV infections in Upper Austrian blood donors as well as a risk estimation of this transfusion-related infection.

Methods and Findings

A total of 58,915 blood donors were tested for HEV RNA using a commercial HEV RT-PCR Kit. 7 of these donors (0.01%) were PCR-positive with normal laboratory parameters in absence of clinical signs of hepatitis. Viral load determined by quantitative real-time PCR showed a HEV nucleic acid concentration of 2,217 293,635 IU/ml. At follow-up testing (2–11 weeks after donation) all blood donors had negative HEV RNA results. Additionally, genotyping was performed by amplification and sequencing of the ORF1 or ORF2 region of the HEV genome. All HEV RNA positive donor samples revealed a genotype 3 isolate. For the antibody screening, anti-HEV IgM and IgG were detected by ELISA. Follow up serological testing revealed that no donor was seropositive for HEV IgM or IgG antibodies at time of donation. Moreover, we verified the prevalence of anti-HEV IgG in 1,203 of the HEV RNA negative tested blood donors. Overall 13.55% showed positive results for anti-HEV IgG.

Conclusions

In the presented study, we investigated HEV infections in blood donations of Upper Austria over 1 year. We concluded that 1 out of 8,416 blood donations is HEV RNA positive. Seroprevalence of anti HEV IgG results in an age-related increase of 13.55%. Therefore, based on this data, we recommend HEV-PCR screening to prevent transmission of hepatitis E virus by transfusion.  相似文献   

13.
Abstract Hepatitis E virus (HEV) is the causative agent of non-A, non-B hepatitis which is transmitted by the fecal-oral route and occurs principally in the form of large epidemics and outbreaks in developing countries. Two overlapping synthetic peptides corresponding to overlapping DNA sequences of the ORF 3 of HEV genome were found to be immunoreactive with sera from patients involved in two epidemics of enterically transmitted non-A, non-B hepatitis. The results suggested the existence of two distinct epitopes. The four synthetic peptides representing these two epitopes from Burma and Mexico strains of hepatitis E virus, were used to investigate anti-HEV reactivities. HEV antibodies were detected in 84–88% of HEV-infected individuals according to the peptide used. The results suggest that a peptide-based ELISA can provide an accurate tool for the diagnosis of acute hepatitis type E.  相似文献   

14.
Hantaviral antibodies were detected in the sera from patients with hepatic disease of unknown etiology in Japan by several different serological diagnostic methods. A total of 105 sera from diseased patients which were negative to A-G hepatitis virus infections in the Tokyo area were tested. Among them, 3 out of 73 sera from patients with chronic hepatic disease were positive to hantaviral antibody by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent antibody assay (IFA) and Western blot analysis (WB). Neutralizing antibody titers of the 3 sera to Seoul virus (SEO) were 4 to 8 times higher than those to Hantaan virus (HTN). However, all of the 32 sera from patients with acute hepatitis were negative for hantaviral antibody. Among the 60 patients with chronic hepatitis in Hokkaido which were serologically negative to B and C hepatitis virus infection, one was positive for hantaviral antibody by ELISA and WB. In contrast, the sera from healthy adults in Japan, 550 from the Honshu and Kyushu regions, and 1,000 from the Hokkaido region, were negative for hantavirus antibody. These results show that hantaviral antibodies are more frequently detected in patients with hepatic disease than in healthy adults. However, the observation that no positive sera were detected from patients with acute hepatitis implies that hantavirus might not be directly related to hepatitis.  相似文献   

15.
戊型肝炎病毒(HEV)合成肽及基因重组抗原免疫反应性研究   总被引:2,自引:0,他引:2  
用ORF2、ORF3合成肽抗原(1~10号)及基因工程重组的ORF2抗原(1和2号)分别建立了酶联免疫方法(EIA),检测60份戊型肝炎病人血清中HEVIgG及IgM10个合成肽抗原(Sp1-Sp10)及2个重组抗原(Re1、Re2),均和HEV阳性血清发生特异反应,但阳性率和反应强度差别很大。以Re1(ORF2,402~660)检测的抗体阳性率最高,为96.7%(58/60);Sp6(ORF3,88~123)次之,为93.3%(56/60);以上两种抗原混合使用阳性率为100%(60/60)。Sp6、Re1及这两种抗原混合使用检测抗HEVIgM,阳性率分别为18.3%(11/60)、66.7%(40/60)和66.7%(40/60)。研究结果表明:合成肽6号(Sp6)及重组抗原1号(Re1)是制备戊肝抗体诊断试剂的理想抗原。  相似文献   

16.
Antibody to hepatitis B virus core antigen (anti-HBc) is one of the most important serological markers during hepatitis B virus (HBV) infection. The quality of the hepatitis B virus core antigen (HBcAg; diagnostic antigen) is crucial to the accuracy of anti-HBc detection. In an attempt to explore the suitability of recombinant HBcAg (rHBcAg) for diagnostic purposes, HBcAg was expressed in Escherichia coli (E. coli) and Pichia pastoris (P. pastoris) and evaluated for the detection of anti-HBc. The expression level of the recombinant protein satisfied the criteria for large-scale biologic production. P. pastoris- and E. coli-derived rHBcAg were purified with gel filtration followed by sucrose gradient (reagents A and C) or with a monoclonal anti-HBc antibody binding (reagents B and D) and were utilized to detect anti-HBc in competitive inhibition enzyme-linked immunosorbent assay (ELISA) format. The ELISA using P. pastoris-derived rHBcAg had a higher specificity and sensitivity than that using E.coli-derived rHBcAg to detect the anti-HBc standard panel. Serum specimens were collected from HBV-infected patients and healthy individuals (voluntary blood donors). Anti-HBc was detected in those specimens using P. pastoris- and E. coli-derived rHBcAg. The positive rate of anti-HBc detection in HBV-infected patients' sera was 100% with reagents A and B, 96.4% with reagent C, and 93.6% with reagent D. The negative rate in healthy control sera was 100% with reagents A and B, 97.0% with reagent C, and 99.7% with reagent D. These data indicate that P. pastoris-derived rHBcAg is superior to E.coli-derived rHBcAg for the detection of anti-HBc using the diagnostic ELISA.  相似文献   

17.
丙型肝炎病毒(Hepatis C virus,HCV)感染者的血液中病毒含量极低,还没有适当的方法可以直接检测病毒。现在常用免疫学方法测定特异性抗体[1]或利用 PCR 技术检测病毒核酸。由于 HCV基因组变异高达33%,PCR检测易发生假阳性和假阴性。同时由于丙肝抗原高度的变异性[2],虽然目前ELISA方法应用广泛,但是单一的诊断抗原已不能满足临床要求,需要多价抗原联合应用作为诊断试剂。目前,在我国流行的丙型肝炎病毒主要是Ⅱ型和Ⅲ型,国外的丙型肝炎诊断试剂研制单位主要是以Ⅰ型为主[3],而且进口的丙型肝炎诊断试剂昂贵,不适合在我国大量使…  相似文献   

18.
Porcine circovirus type 2 (PCV2) is considered to be associated with post-weaning multisystemic wasting syndrome (PMWS), which is a newly emerged economically important swine disease. The entire coding region of open reading frame 2 (ORF2), encoding the viral capsid protein (Cap), of PCV2 was cloned and sequenced from the clinical specimen obtained from PMWS-affected piglets. Six recombinant subunits, A-F, spanning the defined regions of Cap were produced by E. coli expression system and used as antigens for testing their reactivities with swine sera in the indirect ELISA. The recombinant Cap subunit-based ELISA was evaluated by examining a panel of 12 PCV2-negative and 26 PCV2-positive sera. When the positive/negative cut-off value was set at the mean value of negative sera plus 3 standard deviations, all subunits-based ELISA demonstrated 100% specificities. The N-terminal subunits, A and B, revealed poor reactivity with positive swine sera, whereas, greater immunoreactivity was observed for the C-terminal subunits of which subunits C and D demonstrated good sensitivities of 96.2% and 84.6%, respectively. The recombinant Cap subunits possessing defined antigenicity are easy to produce and the subunit-based ELISA was developed with a high specificity and sensitivity that may provide a useful method for routine serodiagnosis of PCV2 infection.  相似文献   

19.
In laboratory animal facilities, monkeys and pigs are used for animal experiments, but the details of hepatitis E virus (HEV) infection in these animals are unknown. The risk of infection from laboratory animals to humans has become a concern; therefore, much attention should be paid to the handling of these animals during their care and use, including surgical procedures performed on infected animals. In this connection, serum samples collected from 916 monkeys and 77 pigs kept in 23 animal facilities belonging to the Japanese Association of Laboratory Animal Facilities of National University Corporations (JALAN) and the Japanese Association of Laboratory Animal Facilities of Public and Private Universities (JALAP) in Japan were examined for the purpose of detecting antibodies to HEV and HEV RNA by using ELISA and RT-PCR, respectively. One hundred and seven serum samples of 916 (11.7%) monkeys were positive for anti-HEV IgG, and 7 and 17 serum samples of 916 (0.8% and 5.3%) monkeys were positive for anti-HEV IgM and IgA, respectively. Thirty-six samples from 62 (58.1%) farm pigs were positive for anti-HEV IgG, whereas all samples tested from miniature pigs were negative (0/15, 0%). Seven samples from 62 (9.1%) farm pigs and 7 samples from 916 (0.8%) monkeys were positive for IgM antibody, but these HEV-IgM antibody positive serum samples were HEV-RNA negative by RT-PCR. The IgM antibody positive rate (9.1%) of farm pigs was much higher than that of monkeys (0.8%). These results suggest the relative levels of risk of HEV infection from these animals to animal handlers and researchers who work with them in laboratory animal facilities.  相似文献   

20.
Hepatitis A and hepatitis E are enteric transmitted viral diseases occurring in epidemic and sporadic forms especially in developing countries. Previous studies in Turkey showed that most residents are infected with HAV by the second decade of life. Since HEV is generally transmitted by the same route as HAV we conducted a community-based seroprevalence study for HAV and HEV infection in Ahatli area in Antalya, Turkey where socioeconomic conditions are low. Anti-HAV total immunoglobulin was tested by using a microparticle EIA (Axsym-Abbott Lab). Anti-HEV IgG was assayed by a micro ELISA method (Genelabs-Singapore). Of the 338 sera tested, 112 (33.1%) were positive for anti-HAV total antibody. Anti-HEV IgG was detected in three (0.89%) of the serum samples. Seropositivity rates of HAV in preschool and school children were 19.9% and 43.9% respectively (p < 0.001). No antibody to HEV was detected in preschool children, while the prevalence of anti-HEV IgG was 1.6% in children attending school. Our data showed that seroprevalence of anti-HAV is high among children samples but HEV infection appears to be relatively rare in pediatric age groups.  相似文献   

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