乙型肝炎表面抗原疑难判定血清的研究

用中国药品生物制品检定所检定合格的国内外HBsAg EIA试剂及ABBOTT抗-HBs、抗-HBc EIA试剂,对所收集的13份HBsAg疑难判定血清进行检测,并用PCR方法检测血清中HBV DNA,结果显示国内外HBsAg试剂对部分HBV DNA阳性样品的检出率差异较大,提示这些样品可能为S基因突变株或HBsAg含量较低,因此,HBsAg EIA试剂的敏感度仍有待进一步提高,并应进一步研制检测S基因突变株的HBsAg诊断试剂。     

乙肝病毒表面抗原和抗体双阳性者中病毒S区基因序列分析

为了解乙肝病毒 (HBV)表面抗原和抗体双阳性患者中病毒的基因型及其HVBS区是否有变异。用放射免疫试剂检测HBsAg阳性样品中的抗 HBs抗体 ,用聚合酶链反应法检测双阳性样品中的HBVDNA ,然后对阳性样品进行克隆和基因序列分析 ,并将所得序列与HBV不同基因型的代表株进行比较分析。结果显示 389例HBsAg阳性样品中有 10例为抗HBs抗体阳性 ;该 10例双阳性样品中有 5例为HBVDNA阳性 ;序列分析显示该 5株HBV均为B基因型 ,其中 4株为adw亚型 ,1株为adr亚型 ;其中有 2株在S区的“a”决定簇的氨基酸发生了变异     

乙型肝炎表面抗原凝难判定血清的研究

用中国药品生物品检定所检定合格的国内外ＨＢｓＡｇＥＩＡ试剂及ＡＢＢＯＴＴ抗－ＨＢｓ、抗－ＨＢｃＥＩＡ试剂,对所收集的１３份ＨＢｓＡｇ疑难判定血清进行检测,并用ＰＣＲ方法检测血清中ＨＢＶＤＮＡ,结果显示国内外ＨＢｓＡｇ试剂对部分ＨＢＶＤＮＡ阳性样品的检出率差异较大,提示这些样品可能为Ｓ基因突变株或ＨＢｓＡｇ含量较低,因此,ＨＢｓＡｇＥＩＡ试剂的敏感度仍有待进一步提高,并应进一步研制检测Ｓ基因突变株的     

乙肝病毒表面抗原和抗体双阳性者中病毒S区基因序列分析

为了解乙肝病毒(HBV)表面抗原和抗体双阳性患者中病毒的基因型及其HVB S区是否有变异.用放射免疫试剂检测HBsAg阳性样品中的抗-HBs抗体,用聚合酶链反应法检测双阳性样品中的HBV DNA,然后对阳性样品进行克隆和基因序列分析,并将所得序列与HBV不同基因型的代表株进行比较分析.结果显示389例HBsAg阳性样品中有10例为抗HBs抗体阳性;该10例双阳性样品中有5例为HBV DNA阳性;序列分析显示该5株HBV均为B基因型,其中4株为adw亚型,1株为adr亚型;其中有2株在S区的"a"决定簇的氨基酸发生了变异.     

乙肝病毒表面抗原的抗体双阳性者中病毒S区基因序列分析

为了解乙肝病毒（HBV）表面抗原和抗体双阳性患者中病毒的基因型及其HVB S区是否有变异。用放射免疫试剂检测HBsAg阳性样品中的抗－HBs抗体，用聚合酶链反应法检测双阳性样品中的HBV DNA，然后对阳性样品进行克隆和基因序列分析，并将所得序列与HBV不同基因型的代表株进行比较分析。结果显示389例HBsAg阳性样品中有10例为抗HBs抗体阳性；该10例双阳性样品中有5例为HBV DNA阳性；序列分析显示该5株HBV均为B基因型，其中4株为adw亚型，1株为adr亚型；其中有2株在S区的“a”决定簇的氨基酸发生了变异。     

一种联合检测乙型肝炎病毒前S1抗原与核心抗原方法的建立及其与病毒核酸检测结果的一致性

本研究以与血清中HBV DNA含量高度相关的两种HBV抗原(前S1抗原与核心抗原)为靶标,建立了联合检测这两种HBV核酸相关抗原(NRAg)的双抗体夹心法ELISA试剂.对系列稀释血清的检测表明,该试剂的平均分析灵敏度为103.2基因组拷贝/mL(95%可信限102.2-4.2基因组拷贝/mL),显著高于前S1抗原或核心抗原的单独检测.对994份HBsAg阴性血清的检测结果表明NRAg ELISA的特异性为99.7%(95%可信限:99.1%～99.9%).对271份临床慢性肝炎血清进行检测,结果NRAg ELISA与HBV DNA结果的总符合率达96.3%(95%可信限:93.3%～98.2%),NRAg ELISA的读值/临界值比(S/CO)与HBV基因组拷贝数呈正相关.利用NRAg试剂,发现了1例HBsAg"a"抗原表位突变的变异株.这些结果显示HBV NRAg ELISA与HBV DNA具有高度相关性,并能够检测出HBsAg抗原变异株,有望成为HBsAg变异株筛选的有力工具,并为广大基层医疗单位提供一种便捷的替代HBV DNA定性检测的手段.     

国产放射免疫试剂与Abbott放射免疫试剂检测乙型肝炎病毒感染指征的比较

本文报告了国产乙型肝炎病毒(HBV)RIA和Abbott RIA诊断试剂盒检测质量的比较研究。表明两试剂滴定HBsAg、抗-HBs、抗-HBc阳性血清的敏感性相似,抗-HBs mIU/ml定量检测结果相符。证明国产RIA试剂质量是可靠的。但是,国产试剂S/N值曲线的陡度和标本的阳性检出率较Abbott试剂为高,可能与试剂出厂后周转期短有关。适当提高国产试剂阳性判定标准后,与Abbott试剂的检测符合率可达90％以上。     

抗乙型肝炎病毒表面抗原单克隆抗体的研究Ⅱ．抗HBsAg单克隆抗体的实际应用

本文用抗HBs／a McAb腹水用亲和层析法有效地提纯了HBsAg,回收率较高,并进行了CIEP检测临床血清标本,沉淀线清晰,检测灵敏度高于抗HBsAg血清。将抗HBs／a和抗HBs／d二种McAb提纯后制备诊断血球,进行RPHA检测临床血清标本千余例,并经美国Abbott实验室RIA试剂盒验证,表明其阳性检出率与中和试验符合率较国内目前以PcAb制备的诊断血球为优,显示了良好的特异性、灵敏度和准确性。抗HBsAgMcAb可作为新一代的HBsAg检测试剂。     

乙型肝炎病毒前S1抗原与HBV血清标志物同时检测及临床意义

目的 :了解前S1抗原与HBV血清标志物的关系。方法 :采用ELSIA方法同时检测 2 1 0 1例乙型肝炎病毒感染者的前S1抗原和HBV血清标志物。结果 :2 9例HBsAg( )、HBeAg( )标本中 ,有 2 8例前S1抗原阳性 ,阳性率 96.5 5 % ;784例HBsAg( )、HBeAg( )、抗HBc( )标本中 ,有 679例前S1抗原阳性 ,阳性率 86.61 % ;2 76例HBsAg( )、抗HBc( )标本中 ,有 1 97例前S1抗原阳性 ,阳性率 71 .38% ;1 0 1 2例HBsAg( )、抗Hbe( )、抗HBc( )标本中 ,有 468例前S1抗原阳性 ,阳性率 46.2 5 %。结论 :前S1抗原作为病毒复制的指标与HBeAg具有很好的一致性 ,又具有其独立的检测价值 ,可弥补HBV血清标志物检测的不足。     

HBV感染中抗-(抗-HBs)独特型抗体的研究

1.75％聚乙二醇(MW6000)沉淀血清免疫复合物后,采用ELISA法检测上清中抗独特型抗体〔抗-(抗-HBs)〕。56例急性HBV感染患者血清IgM抗-(抗-HBs),第一周检出率最高(60.00％),1个月后大部分阴性,IgG抗-(抗-HBs)维持时间略长。IgM抗-(抗-HBs)较HBsAg/IgM和IgM抗-HBc消失早,将有利于急性HBV感染的诊断。27例CAH和29例CPH,IgM和IgG抗-(抗-HBs)阳性率约为38～52％,两型间无显著差异(p>0.5)。3例无症状HBsAg携带者和12例HBsAg血清疫苗接受者血清,抗-(抗-HBs)阴性。115例HBV感染者中,IgM抗-(抗-HBs)阳性者HBsAg阳性率(93.62)显著高于IgM抗-(抗-HBs)阴性者(52.31％),p<0.005;反之,HBsAg阳性血清IgM抗-(抗-HBs)阳性率(55.5S％)显著高于HBsAg阴性血清(9.68％),p<0.005;IgM抗-(抗-HBs)阳性和阴性血清的HBVDNA阳性率有显著差异(p<0.025),IgG反应到类似的结果。以上资料表明,抗-(抗-HBs)提示了一些HBV感染患者体内可能存在一种缺陷的反馈机制,导致抗-HBs产生不足而容许更活跃的HBV复制。本文证明了HBV感染患者血清中存在的抗-(抗-HBs)可能是共同决定簇“a”特异性的,还表明抗-HBs人抗-Id反应可能被纯化人血清HBsAg抑制,提示人抗-(抗-HBs)与抗-HBs结合的位点可能在HBsAg与抗-HBs结合位点内或在其附近,这和一些动物抗-(抗-HBs)的研究结果是一致的。     

Rare Detection of Occult Hepatitis B Virus Infection in Children of Mothers with Positive Hepatitis B Surface Antigen

The prevalence of occult Hepatitis B virus (HBV) infection in children was considerably varied from 0.1–64% in different reports. In this study we aimed to investigate the prevalence of occult HBV infection among the children born to mothers with positive hepatitis B surface antigen (HBsAg) in Jiangsu, China. Serum samples were collected from 210 children of 207 mothers with positive HBsAg. HBV serological markers were detected by ELISA and HBV DNA was detected by nested PCR. Homology comparison of HBV sequences recovered from the child and mother was used to define the infection. Three children (1.43%) were positive for HBsAg, in whom the HBV pre S and S gene sequence in each child was identical to that in her mother. Of the 207 HBsAg-negative children, nine displayed HBV DNA positive by two nested PCR assays using primers derived from S and C genes. However, the sequence alignment showed that the sequences in each child were considerably different from those in his/her mother. Therefore, the sequences amplified from the children were very likely resultant from the cross-contaminations. Furthermore, the nine children with ‘positive HBV DNA’ were all negative for anti-HBc, and one had anti-HBs 3.42 mIU/ml and eight others had anti-HBs from 72 to >1000 mIU/ml, indicating that the nine children were less likely infected with HBV. Therefore, none of the 207 HBsAg-negative children of HBV-infected mothers was found to have occult HBV infection. We conclude that the prevalence of occult HBV infection in vaccinated children born to HBsAg positive mothers should be extremely low. We recommend that homology comparison of sequences recovered from the child and mother be used to define the occult HBV infection in children born to HBV infected mothers.     

Vaccine- and hepatitis B immune globulin-induced escape mutations of hepatitis B virus surface antigen

Hepatitis B virus surface antigen (HBsAg) vaccination has been shown to be effective in preventing hepatitis B virus (HBV) infection. The protection is based on the induction of anti-HBs antibodies against a major cluster of antigenic epitopes of HBsAg, defined as the 'a' determinant region of small HBsAg. Prophylaxis of recurrent HBV infection in patients who have undergone liver transplantation for hepatitis B-related end-stage liver disease is achieved by the administration of hepatitis B immune globulins (HBIg) derived from HBsAg-vaccinated subjects. The anti-HBs-mediated immune pressure on HBV, however, seems to go along with the emergence and/or selection of immune escape HBV mutants that enable viral persistence in spite of adequate antibody titers. These HBsAg escape mutants harbor single or double point mutations that may significantly alter the immunological characteristics of HBsAg. Most escape mutations that influence HBsAg recognition by anti-HBs antibodies are located in the second 'a' determinant loop. Notably, HBsAg with an arginine replacement for glycine at amino acid 145 is considered the quintessential immune escape mutant because it has been isolated consistently in clinical samples of HBIg-treated individuals and vaccinated infants of chronically infected mothers. Direct binding studies with monoclonal antibodies demonstrated a more dramatic impact of this mutation on anti-HBs antibody recognition, compared with other point mutations in this antigenic domain. The clinical and epidemiological significance of these emerging HBsAg mutants will be a matter of research for years to come, especially as data available so far document that these mutants are viable and infectious strains. Strategies for vaccination programs and posttransplantation prophylaxis of recurrent hepatitis need to be developed that may prevent immune escape mutant HBV from spreading and to prevent these strains from becoming dominant during the next decennia.     

Genetic variability of hepatitis B virus isolates among population of Shuryskarsky area of Yamal-Nenets autonomous region

The purpose of this work was to determine occurrence of serological markers of hepatites B and to describe subtypes of a superficial antigen and genotypes of hepatitis B virus (HBV) isolates among indigenous population of Yamal-Nenets Autonomous Region (YNAR), Russia. METHODS: We investigated 657 serum samples from inhabitants of Shuryskarsky area of YNAR. ELISA method was used to define the hepatitis B markers: HBsAg, anti-HBs (total) and anti-HBc (IgG and IgM). The HBsAg-positive samples were PCR-tested for the presence of HBV DNA. Genotyping of isolates was by sequencing of the Pre-Sl/Pre-82/S region of HBV genome and phylogenetic analysis. Definition of HBsAg subtypes was executed by two methods: ELISA with subtype-specific monoclonal antibodies and S-gene nucleotide sequence analysis. RESULTS: The following occurrence of hepatitis B markers was observed: HBsAg - 3.2%, anti-HBs (total) - 36.2%, anti-HBc IgG - 30.3%, anti-HBc IgM - 1.6%. Frequency of carrying even one of the markers in the observed population was 47.5%. HBV DNA was found in 17 HBsAg-positive samples. Pre-SI, Pre-S2 and S regions sequences were determined for all HBV DNA-positive samples. The phylogenetic analysis showed an accessory of all investigated HBV isolates to genotype D. HBsAg subtypes distribution appeared the following: ayw2 - 23.5%, ayw3 - 70.6%, adw2 - 5.9%. Results of definition of the subtype ELISA method and by the analysis of S gene nucleotide sequences have coincided in 10/11 (90.1%) cases. CONCLUSIONS: The indigenous population of Shuryskarsky area of YNAR belongs to groups with average HBV carrying. Absolute domination of genotype D (subtypes ayw2, ayw3 and adw2) was revealed. High percentage of concurrence of HBsAg subtypes detected by the ELISA method and method of the analysis of S gene primary structure (90%) was observed. Sequencing of HBV S-gene is preferable to define HBsAg subtypes.     

慢性乙型肝炎病毒耐药突变体研究

在治疗慢性乙型肝炎的核苷类似物的用药过程中,筛选耐药突变体.选定1名从未接受抗病毒治疗慢性乙型肝炎患者,用抗病毒核酸类药物治疗,不同治疗时期提取血清HBV DNA,用引物P1(5′-AAGGG-TATCTTGCCCGTTTGTCGTA-3′)和P2(5′-AAGCAGGATAGCCACAGA-3′)为第1轮,P3(5′-AAGGCACTTGTAT-TCCCATCCGAG-3′)和P4(5′-AAGGTCTATTTACAGGGGA-3′)为第2轮引物扩增筛选耐药突变体.在18周和22周分别检测到突变体LMV rtH69T(YMDDlocus)和LMV rtT184T(YMDDlocus)、LMV rtM204I(YMDDlocus).在60周和70周分别检测到ADV T213S、ADV T222A、ADV K212T和ADV S196L、ADV S242H,其中ADV S196L和ADV S242H 2种突变体是首次检测到.HBV核苷酸类似物耐药突变体筛选,对研究HBV耐药的分子机制有帮助.     

乙型肝炎病毒DNA在患者血清及肝组织中存在状态的研究

对29例肝炎,1例尸检肝组织和血清中乙型肝炎病毒的DNA(HBV DNA)进行了研究,发现HBsAg( )/HBeAg( )患者中,有9/17(52.94％)血清HBV DNA阳性;HBsAg( )/抗-HBe( )患者中,2/6(33.33％)也为阳性。从30例肝组织中提取DNA经琼脂糖电泳,Southern吸印转移及分子杂交试验结果表明,27例HBV DNA阳性,全部有游离型HBV DNA。27例中有5例经用标记pBR322探针杂交排除非特异杂交带后,在高分子量区有HBV DNA特异的杂交带,提示有HBV DNA整合。     

Challenges in the Testing of Non-Heart-Beating Cadavers for Viral Markers: Implications for the Safety of Tissue Donors

Natural changes that occur in blood and tissue after death may result in false positive results in antigen and antibody detection tests performed to identify markers of viral infection in potential tissue donors. Such tissue, which might otherwise be acceptable for therapeutic purposes, would not meet current standards for safe tissue banking. This is especially important in the context of insufficiency in the tissue supply. In this study, a series of blood samples collected during routine post-mortem examination was assayed using a range of commercially available kits for the detection of HBsAg, anti-HCV and anti-HIV 1 + 2 antibody/antigen. Results of tests on 104 samples collected from 97 individuals indicate that some kits result in a higher number of initial reactive samples than others. Approximately 40% of samples were reactive in one or more HBsAg assay, less than 10% in at least one anti-HIV kit and only 1 sample at low level on an anti-HCV kit. Liver or lymph node samples from individuals whose serum sample gave reactive results in antigen/antibody assays were tested for viral nucleic acid in the corresponding nucleic acid amplification test. Only one individual’s sample was confirmed to test positive for HBsAg in a confirmatory neutralisation test and by nucleic acid amplification technology, and a second individual whose serum was scored reactive for anti-HCV, but negative for HBsAg, had a liver sample which was HBV DNA positive and HCV RNA negative. The results of the study indicate that antibody/antigen assays are not as specific as NAT using state of the art DNA extraction techniques. Both types of assay complement each other and used together will help assure the safety of tissues for transplantation.     

硫代磷酸反义寡脱氧核苷酸抗HBV细胞的实验研究

针对ＨＢＶ的５个基因位点作为靶序列,设计合成硫代反义寡聚核苷酸（Ｓ－ａｓＯＤＮ）．应用ＥＬＩＳＡ方法、ＭＴＴ比色法、电子显微镜等手段,观察Ｓ－ａｓＯＤＮ对ＨｅｐＧ２２２１５细胞ＨＢｓＡｇ、ＨＢｅＡｇ抗原的表达,及细胞的毒性、细胞形态和超微结构的影响．结果显示不同靶位点的Ｓ－ａｓＯＤＮ对ＨＢｓＡｇ、ＨＢｅＡｇ表达都有显著的抑制作用,且表现为序列特异性、剂量相关性、联合协同性和一定的抗核酸酶性,在浓度为２０μｍｏｌ／Ｌ时,对细胞无明显杀伤作用,对细胞的超微结构无显著的改变．结果提示Ｓ－ａｓＯＤＮ有望发展为抗ＨＢＶ的有效药物,但靶序列的选择、透细胞膜性及联合用药配伍等仍值得进一步研究和解决．     

Clinical and Virological Characteristics of Chronic Hepatitis B Patients with Coexistence of HBsAg and Anti-HBs

Coexistence of hepatitis B surface antigen (HBsAg) and antibody against HBsAg (anti-HBs) comprises an atypical serological profile in patients with chronic hepatitis B virus (HBV) infection. In this study, in total 94 patients with coexisting HBsAg and anti-HBs and 94 age- and sex-matched patients with positive HBsAg were characterized by quantitatively measuring HBsAg and HBV DNA, sequencing large S genes, and observing clinical features. Compared with common hepatitis B patients, the patients with coexisting HBsAg and anti-HBs had lower HBsAg and HBV DNA levels. These two groups had similar rate of pre-S deletion mutations. However, in patients with coexisting HBsAg and anti-HBs, more amino acid substitutions in the a determinant of S gene were observed in HBV genotype C, but not in genotype B. Fourteen patients with coexisting HBsAg and anti-HBs were followed up for an average of 15.5 months. There were no significant changes in the levels of HBsAg, anti-HBs, HBV DNA and ALT over the follow-up period. Compared with the baseline sequences, amino acid substitutions in the MHR of HBsAg occurred in 14.3% (2/14) patients. In conclusion, coexistence of HBsAg and anti-HBs may be associated with higher frequency of mutations in the a determinant of HBV genotype C.