LightCycler实时监测PCR定量分析血清HBV DNA

目的 检验LightCycler实时监测PCR（real-time detection PCR,RTD-PCR)对血清中HBV DNA定量检测的灵敏性和可重复性，探讨HBV血清标志物与HBV DNA定量的关系。方法 HBV定量按深圳匹基公司乙肝PCR荧光检测试剂盒使用说明，对乙肝标志物已明确的773例血清中HBV DNA定量结果进行统计分析。结果 实时监测PCR对血清中HBV DNA定量检测的灵敏性高，可检测低至1000拷贝／ml血清；可重复性好，批间误差＜20％；各种标志物类型的血清HBV DNA含量分布情况表明，HBV血清标志物中HBeAg与HBV DNA含量有明显的关系，一般HBeAg阳性血清HBV DNA含量较高，但也有相当一部分例外。结论 LightCycler实时监测PCR对血清中HBV DNA定量检测灵敏性高可重复性好；仅根据HBV血清标志物往往不能确定乙肝患者HBV DNA复制水平的高低，HBV DNA定量检测具有十分重要的临床意义。     

紫丁香叶提取物对HepG2.2.15细胞中HBeAg及HBsAg表达的影响

目的 :观察紫丁香叶提取物对 Hep G2 .2 .1 5细胞分泌 HBe Ag和 HBs Ag的影响。方法 :采用酶联免疫吸附法 ( ELISA)检测培养上清中 HBe Ag和 HBs Ag表达的变化并以抑制率来表示。结果 :紫丁香叶提取物对 HBe Ag和 HBs Ag的分泌具有抑制作用 ,该作用呈现剂量依赖性和时间依赖性。结论 :紫丁香叶提取物对 HBe Ag和 HBs Ag分泌的抑制作用提示其在体外具有抗乙型肝炎病毒的作用     

生物素－链霉亲和素系统在检测HBeAg中的比较

应用链霉亲和素和生物素系统发展成的酶连接免疫吸附测定（ＥＬＩＳＡ）改良法检测ＨＢｅＡｇ，取得较满意效果。标记链霉亲和素－辣根过氧化物酶（ＳＡ－ＨＲＰ）时，两者的重量比以（０．５～０．８）：１为佳，浓度以７μｇ／ｍｌ为最理想；ＳＡ－ＨＲＰ的反应时间以３０ｍｉｎ为适宜。本改良法与生物素亲和素ＥＬＩＳＡ（ＢＡ）法、普通ＥＬＩＳＡ法检测ＨＢｅＡｇ的灵敏度比较，其相对灵敏度高于后二者；用Ａｂｂｏｔｔ试剂作为标准，检测ＨＢｅＡｇ１００人份，其阳性率高于ＢＡ法和普通ＥＬＩＳＡ法。提示链霉亲和素ＥＩ，ＩＳＡ（ＢＳＡ）法可提高方法的特异性及灵敏度。     

阿德福韦酯联合辨证使用中药治疗HBeAg阳性慢性乙肝的临床研究

目的：研究阿德福韦酯联合辨证使用中药对HBeAg阳性慢性乙肝患者的实验室指标改善情况。方法：选择120例HBeAg阳性的慢性乙肝患者,随机分2组各60例接受治疗,2组病例均每日口服阿德福韦酯10mg,治疗组联合辨证使用中药,52周后停药。观察第12、52周及第78周ALT水平及病毒学指标方面的改变。结果：12周、78周时,治疗组HBV-DNA降低水平、HBeAg转阴率、HBeAg/HBeAb血清学转换率高于对照组,12周、52周、78周时,ALT复常率均高于对照组,余均无明显差异。结论：阿德福韦酯联合辨证使用中药对于HBeAg阳性慢性乙肝患者病毒抑制具有一定的协同作用以及较好的增强持久应答的作用。     

Hepatitis B virus-DNA in the serum of patients followed-up longitudinally with acute and chronic hepatitis B

Sera from 79 patients with acute self-limiting hepatitis, 17 patients with acute hepatitis B evolving into chronic HBsAg carriership, and 43 chronic HBsAg carriers without a history of acute hepatitis were analyzed for presence of hepatitis B virus (HBV)-DNA by a molecular hybridization technique. In acute self-limiting hepatitis, HBV-DNA was cleared within a few weeks after the onset of clinical symptoms. The longest period of DNA positivity observed in this group was 42 days. In 29 of 52 patients HBV-DNA was cleared before HBeAg disappeared. Among 17 patients who became chronic HBsAg carriers, HBV-DNA was present for more than 6 months in all but one. Most of the HBsAg carriers eventually cleared HBV-DNA. The DNA clearance frequently preceeded the conversion of HBeAg to anti-HBe. Thus, in many patients there was a transitional period with HBeAg but without HBV-DNA. HBV-DNA was found to be a better index of impending chronicity than HBeAg since persistence of HBeAg for more than 42 days was noted in 10% of the patients who nevertheless cleared HBsAg within 6 months. By that time all those patients had turned negative for HBV-DNA. On the other hand, in 16 of the 17 patients who became chronic carriers of HBsAg, HBV-DNA as well as HBeAg persisted for more than 6 months. The present results also suggest that infectivity in acute hepatitis B is a feature mainly of the presymptomatic and early symptomatic period.     

HBeAg时间分辨荧光免疫分析法的建立

采用平衡饱和法建立了乙型肝炎病毒e抗原(HBeAg)时间分辨荧光免疫分析法.以针对HBeAg的单克隆抗体G8包被板,双功能螯合剂异氰酸苄基二乙烯三胺四乙酸络合Eu3+及标记C4单抗,发光增强系统为以β-二酮体为主的增强液.数据采用Log-Logit法函数和四参数Logitc函数数据处理程序处理.结果表明方法的批内和批间CV分别为2.39%和5.28%,平均回收率为97.62%,灵敏度为0.58NCU/mL,可测范围为12.01-529.84NCU/mL,ED20、ED50和ED80分别为6.36NCU/mL、26.85NCU/mL和136.7NCU/mL.本方法与HBsAg有13.1%的交叉反应.Eu3+标记抗体-30℃保存6个月免疫反应性基本无损失,同批试剂连续5个月应用分析结果稳定.HBeAg时间分辨荧光免疫分析的质量参数优于EIA和IRMA.     

Hepatitis B surface antigen particles of subtypes adw and adr, and compound subtype (adwr) in symptom-free carriers in Japan.

Of sera from 1,878 Japanese blood donors who carried hepatitis B surface antigen (HBsAg), 420 were subtyped as adw (22.4%) and 1,443 as adr (76.8%); only 15 (0.8%) contained HBsAg of subtype ayw or ayr. Sera with HBsAg/adr had higher HBsAg titres than those with HBsAg/adw (geometric mean of haemagglutination titre: 10.1 +/- 2.4 vs. 9.7 +/- 2.4, p less than 0.01), and a higher prevalence of hepatitis B e antigen (24% vs. 13%, p less than 0.001). Carriers of HBsAg/adr progressively predominated over those of HBsAg/adw with increasing age. Of sera from 1,863 carriers of HBsAg/adw or HBsAg/adr, 182 (9.8%) contained HBsAg particles with both subtypic determinants in the w/r allele. The presence of w and r determinants on the same particles was ascertained by sandwiching them between monoclonal antibody with the specificity for w and that with the specificity for r. HBsAg particles of compound subtype (adwr) were found more often in sera with hepatitis B e antigen than those without it (145/403 [36.0%] vs. 37/1,460 [2.5%], p less than 0.001). Sera with HBsAg/adwr particles had HBsAg titres higher than those without them (12.4 +/- 1.9 vs. 9.7 +/- 2.3, p less than 0.001). HBsAg/adwr particles arise from phenotypic mixing of the S-gene product of wild-type virus and that of mutants with point mutations for subtypic changes. The results obtained indicated that HBV strains of subtype adr have a higher replicative activity than those of adw, and suggested that mutations in the S gene for subtypic changes would be associated with an active replication of hepatitis B virus.     

Nucleotide mutations associated with hepatitis B e antigen negativity

One hundred and forty four patients with chronic hepatitis B were tested to identify new mutations associated with hepatitis B e antigen (HBeAg) negativity, using a full genome sequence analysis. All the patients were Chinese and had hepatitis B virus infection of genotype C. Patients with none of the pre-core or core promoter mutations were significantly (P < 0.001) less common in the group with anti-HBe (13%) than in the group with HBeAg (56%). The complete nucleotide sequence was determined in four anti-HBe-positive patients who had neither pre-core nor core promoter mutations and in five HBeAg-positive patients who also had neither of these mutations (the groups were matched for age and sex). Six mutations were found to be significantly more common in the former group than in the latter: G529A (3/4 vs. 0/5), C934A (4/4 vs. 1/5), A1053G (4/4 vs. 1/5), G1915T/A (4/4 vs. 0/5), T2005C/A (4/4 vs. 0/5), and C3026T (3/4 vs. 0/5). Three of the six mutations were significantly more common in the four anti-HBe-positive patients who had neither pre-core nor core promoter mutations, compared to 11 HBeAg-positive patients who had pre-core and core promoter mutations, and also compared to 15 anti-HBe-positive patients who had pre-core and core promoter mutations, suggesting further the specificity of these mutations. Of the six mutations, two resulted in amino acid substitution in the polymerase protein, and one is located near the enhancer I region. The results suggest that the six newly discovered mutations are associated with HBeAg negativity.     

Isolation and characterization of liver-derived hepatitis B e antigen

Two subpopulations of hepatitis B e antigen (HBeAg) were isolated from a human liver infected with hepatitis B virus. HBeAg extracted from liver homogenate subsequent to treatment with buffered 3 M NaSCN or 0.5 M MgCl2 banded at the density of 1.13 g/cm3 in CsCl and was polydispersed on gel filtration. In contrast, HBeAg released with phosphate-buffered saline (PBS) was detected mainly at a density of 1.20 g/cm3 in a CsCl gradient and consisted of low molecular weight species on gel chromatography. Polypeptides of 40,000 and 45,000 daltons were found in NaSCN and PBS-released HBeAg preparations, respectively. The results are interpreted as suggestive that liver HBeAg is a dimer of the major core particle polypeptide in different physicochemical forms.