Enhanced Detection of Australia Antigen in Serum Hepatitis Patients by Discontinuous Counter-Immunoelectrophoresis

The use of discontinuous counter-immunoelectrophoresis enhanced the reaction between Au/SH antigen and its antibody in agarose. The ionic strength of the Veronal buffer used in the agarose was 0.015 mu, whereas 0.075 mu Veronal (both pH 8.6) was used for anode and cathode buffers. Electroendosmosis is increased under such conditions. Au/SH antigen and antibody reacted to give sharp lines within 30 to 45 min as compared with conventional counter-immunoelectrophoresis which required 1 to 3 hr or longer.     

High Hepatitis B Surface Antigen Levels Predict Insignificant Fibrosis in Hepatitis B e Antigen Positive Chronic Hepatitis B

Introduction

There is no data on the relationship between hepatitis B surface antigen (HBsAg) levels and liver fibrosis in hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B (CHB).

Methods

Serum HBsAg and HBV DNA levels in HBeAg-positive CHB patients with liver biopsies were analyzed. The upper limit of normal (ULN) of alanine aminotransferase (ALT) was 30 and 19 U/L for men and women respectively. Histologic assessment was based on Ishak fibrosis staging for fibrosis and Knodell histologic activity index (HAI) for necroinflammation.

Results

140 patients (65% male, median age 32.7 years) were recruited. 56 (40%) had ALT ≤2×ULN. 72 (51.4%) and 42 (30%) had fibrosis score ≤1 and necroinflammation grading ≤4 respectively. Patients with fibrosis score ≤1, when compared to patients with fibrosis score >1, had significantly higher median HBsAg levels (50,320 and 7,820 IU/mL respectively, p<0.001). Among patients with ALT ≤2×ULN, serum HBsAg levels achieved an area under receiver operating characteristic curve of 0.869 in predicting fibrosis score ≤1. HBsAg levels did not accurately predict necroinflammation score. HBsAg ≥25,000 IU/mL was independently associated with fibrosis score ≤1 (p = 0.025, odds ratio 9.042).Using this cut-off HBsAg level in patients with ALT ≤2×ULN, positive and negative predictive values for predicting fibrosis score ≤1 were 92.7% and 60.0% respectively. HBV DNA levels had no association with liver histology.

Conclusion

Among HBeAg-positive patients with ALT ≤2×ULN, high serum HBsAg levels can accurately predict fibrosis score ≤1, and could potentially influence decisions concerning treatment commencement and reduce the need for liver biopsy.     

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.     

乙型肝炎病毒前S1抗原检测及其临床意义

目的:探讨乙型肝炎病毒前S1抗原与血清标志物之间的关系,了解其临床意义。方法:采用酶联免疫吸附实验检测乙型肝炎病毒血清标志物及前S1抗原,并采用速率法检测血清丙氨酸氨基转移酶。结果:乙肝表面抗原阳性率为9．2%,乙肝表面抗原阳性人群中前S1抗原阳性率为28．6%;乙肝e抗原阳性及阴性人群中前S1抗原阳性率分别为81．1%、13．9%(p＜0．01);前S1抗原阳性人群ALT(49．5U/L)高于前S1抗原阴性人群(43．2U/L,p＜0．01)。结论:前S1抗原与血清标志物e抗原有较高的一致性,是反映病毒复制的良好指标,能较早发现肝损伤。     

Detection of Hepatitis Associated Antigen by the Latex Agglutination Test

Hepatitis associated antigen may be detected quickly and reliably by the latex agglutination test, using antiserum from guinea pigs immunized with the antigen. The latex test has a sensitivity comparable to the counter current immunoelectrophoresis technique.     

Hepatitis B Antigen in Viral Hepatitis in West London

During the first 12 months of a total population survey 249 patients were seen with viral hepatitis. A total of 215 of these were tested for hepatitis B antigen (HB Ag) by radioimmunoassay and 32 (15%) were positive.More than five times as many men (27) as women (5) were HBAg positive and 19 of the men were between the ages of 20 and 39 years. There were only four drug addicts among those tested, two of whom were positive, as were two of the four patients who were tattooed.Sixty out of 86 children (under 15 years) were tested for HBAg and none was positive.     

Hepatitis B Core Antigen: Immunology and Electron Microscopy

TWO DISTINCT VIRAL ANTIGENS ARE ASSOCIATED WITH THE HEPATITIS B VIRUS: the hepatitis B surface antigen (HB(s)Ag, Australia antigen) and the hepatitis B core antigen (HB(c)Ag). HB(s)Ag, purified from the serum of asymptomatic human HB(s)Ag carriers, and HB(c)Ag, purified from the liver of a chimpanzee acutely infected with hepatitis B virus, were examined by serological and immune electron microscopic methods. Antisera raised against HB(s)Ag reacted with the outer, surface component of the Dane particle and with the 20-nm spherical and tubular particles present in HB(s)Ag-positive serum, but not with the internal component of the Dane particle or with purified HB(c)Ag particles. Antisera raised against purified HB(c)Ag particles reacted with the internal component of the Dane particle and with HB(c)Ag, but not with the surface of the Dane particle or with the 20-nm spherical and tubular particles associated with HB(s)Ag. Purified HB(c)Ag particles, 27 nm in diameter, demonstrated distinct subunits. The infectious form of hepatitis B virus appears to be represented by the 42-nm Dane particle composed of a 27-nm nucleocapsid core component (HB(c)Ag) surrounded by an antigenically and morphologically distinct lipoprotein surface component (HB(s)Ag).     

Microtiter Solid-Phase Radioimmunoassay for Hepatitis B Antigen

A micro-solid-phase radioimmunoassay (micro-SPRIA) for hepatitis B antigen (HB Ag) was developed for use with microtiter serological equipment. Radiolabeled immunoglobulin G was prepared from human and animal sera containing hepatitis B antibody (HB Ab); it was not necessary to isolate specific HB Ab by immunochemical means. A micro-SPRIA prepared with guinea pig reagents was approximately as sensitive as the AusRIA radioimmunoassay, but, like the AusRIA test, yielded false positive results. A micro-SPRIA prepared with human reagents was slightly less sensitive but did not yield false positive results. These micro-SPRIA tests offer several advantages, including conservation of reagents, adaptability to other antigen-antibody systems, ease of performance (especially when testing large numbers of specimens), and economy.     

甲型肝炎、乙型肝炎病毒混合抗原的细胞免疫学研究

评价甲型肝炎病毒和乙型肝炎病毒混合抗原对细胞免疫反应的影响。采用小鼠迟发型超敏反应(DTH)、脾脏淋巴细胞增殖实验和淋巴细胞亚型分群实验 ,对甲肝抗原 (HAAg)、乙肝表面抗原 (HBsAg)和甲乙肝混合抗原 (HAAg +HBsAg)进行检测 ,并进行统计学分析。混合抗原没有降低相应单价抗原的各项细胞免疫反应强度 ,且较单一 ,HBsAg表现出了显著的抗原特异性T淋巴细胞和Th2细胞增殖作用 (P <0 .0 5 )。混合抗原表现出良好的细胞免疫反应原性 ,同时可能辅助B细胞 ,增强体液免疫应答能力。     

肝硬化患者乙肝表面抗原的定量检测及意义

目的：探讨HBsAg定量测定在乙肝相关性肝硬化病程中的变化和意义。方法：选择乙肝相关性肝硬化患者60例纳入实验对象,根据2000年9月（西安）第10次全国病毒性肝炎学术会议修订的《病毒性肝炎防治方案》中的诊断标准分为代偿期组和失代偿期组,其中代偿期组35例,失代偿期组25例。另选取20例乙型肝炎病毒携带者作为对照组。应用电化学发光免疫分析法测定患者血清中HBsAg和HBeAg滴度,免疫荧光定量PCR法检测HBVDNA载量。结果：对照组、肝硬化代偿期组和肝硬化失代偿期组HBsAg滴度分别为：2574.73±3252.27COI、5494.35±2129.84COI和6921.25±1957.60COI,三组之间差别均有统计学意义（P〈0.05）。肝硬化代偿期组中,HBsAg滴度与HBVDNA、HBeAg水平呈负相关性（P〈0.05（）r=-0.350;r=-0.514）。肝硬化失代偿期组中,HBsAg滴度与HBVDNA及HBeAg水平均无明显相关性（r=-0.020;r=0.154）。结论：肝硬化失代偿期HBsAg滴度明显高于肝硬化代偿期,代偿期HBsAg滴度高于HBV携带者组,即HBsAg滴度随肝脏疾病进展呈阶梯型递增。肝硬化代偿期,HBsAg滴度与HBVDNA、HBeAg水平呈负相关性,HBsAg水平可以作为评估病毒复制的参考指标。肝硬化失代偿期,HBsAg滴度与HBVDNA和HBeAg无相关性,不能反映病毒复制水平,不能作为评估病毒复制的参考指标。     

肝硬化患者乙肝表面抗原的定量检测及意义

目的:探讨HBsAg定量测定在乙肝相关性肝硬化病程中的变化和意义。方法:选择乙肝相关性肝硬化患者60例纳入实验对象,根据2000年9月(西安)第10次全国病毒性肝炎学术会议修订的《病毒性肝炎防治方案》中的诊断标准分为代偿期组和失代偿期组,其中代偿期组35例,失代偿期组25例。另选取20例乙型肝炎病毒携带者作为对照组。应用电化学发光免疫分析法测定患者血清中HBsAg和HBeAg滴度,免疫荧光定量PCR法检测HBVDNA载量。结果:对照组、肝硬化代偿期组和肝硬化失代偿期组HBsAg滴度分别为:2574.73±3252.27COI、5494.35±2129.84COI和6921.25±1957.60COI,三组之间差别均有统计学意义(P<0.05)。肝硬化代偿期组中,HBsAg滴度与HBVDNA、HBeAg水平呈负相关性(P<0.05()r=-0.350;r=-0.514)。肝硬化失代偿期组中,HBsAg滴度与HBVDNA及HBeAg水平均无明显相关性(r=-0.020;r=0.154)。结论:肝硬化失代偿期HBsAg滴度明显高于肝硬化代偿期,代偿期HBsAg滴度高于HBV携带者组,即HBsAg滴度随肝脏疾病进展呈阶梯型递增。肝硬化代偿期,HBsAg滴度与HBVDNA、HBeAg水平呈负相关性,HBsAg水平可以作为评估病毒复制的参考指标。肝硬化失代偿期,HBsAg滴度与HBVDNA和HBeAg无相关性,不能反映病毒复制水平,不能作为评估病毒复制的参考指标。     

重组乙型肝炎病毒e抗原的研究进展

乙型肝炎e抗原（HBeAg）是由HBV DNA C基因区编码的一种非颗粒性的分泌型核壳蛋白,临床上将其作为判断HBV活动性复制的指标之一。HBeAg是重要的生物原材料,广泛用于HBV感染者血清学检测的相关诊断用品的制备。重组HBeAg的表达和纯化技术较为成熟,已经在各种表达系统中成功地表达了目的蛋白。而影响其表达的关键因素包括前C区重要位点变异、载体选取以及RNA干扰（RNAi）的影响作用等。因此,提高重组HBeAg的表达量和纯度,避免与HBcAg交叉反应才能满足相关诊断制品的要求。高质量重组乙肝e抗原的获取,能为改善相关诊断制品奠定物质基础,为开发新型HBV治疗和预防性疫苗提供了依据,同时也为HBeAb检测方法学的优化提供可能。     

DNA Polymerase Associated with Human Hepatitis B Antigen

DNA polymerase activity was detected in each of eight preparations of concentrated human hepatitis B antigen (HBAg) rich in Dane particles prepared by high-speed centrifugation of antigen-positive human plasma and in none of seven control preparations prepared in the same way from HBAg-negative plasma. The incorporation of (3)H-thymidine-methyl-5'-triphosphate into DNA was dependent on four deoxyribonucleoside triphosphates and MgCl(2). Treatment of the concentrated HBAg preparations with the nonionic detergent Nonidet P-40 (NP40) more than doubled the enzyme activity. Fractionation of the concentrated HBAg preparation in sucrose density gradients after treatment with NP40 revealed that the enzyme activity appeared within the density range of Dane core antigen but at a slightly higher density than the average for core antigen. The only particles observed by electron microscopy in this region of the gradient were typical 28-nm cores, suggesting that the DNA polymerase activity was associated with a subpopulation of cores. No DNA polymerase activity was found in purified 20-nm HBAg particles. The DNA product of the reaction remained associated with the 110S core and was not susceptible to DNase digestion when associated with the core. Inhibition of the reaction by actinomycin D and daunomycin suggested that the reaction was dependent on a DNA template associated with the core.     

Biophysical and Biochemical Heterogeneity of Purified Hepatitis B Antigen

Hepatitis B antigen of the D (a+, d+, y-) subtype was purified from plasma of apparently healthy persons and from hepatitis patients. The original samples contained 20- and 42-nm particles and tubular forms (20-nm diameter). Ultracentrifugation during the purification procedure yielded pellets which were then treated at pH 2.4. Both the large, 42-nm Dane particles and the tubular forms were lost during the acid treatment of the pelleted particles, yielding a preparation containing a mixture of particles approximately 20 and 25 nm in diameter. This difference in size was substantiated in that two distinct molecular weights were calculated from high-speed equilibrium data, 3.6 x 10(6) and 4.5 x 10(6). Further heterogeneity was observed in that hepatitis B antigenic activity was present in purified particles with an isoelectric pH of 4.0 and also in those with a pH of 4.4. No significant differences were observed in the gross amino acid composition of purified antigen obtained from plasma of three different persons. (125)I-labeled, purified antigen was found to contain six distinct polypeptides with molecular weights ranging from 10,000 to 39,000.     

Specificity and Sensitivity of Radioimmunoassay for Hepatitis B Antigen

Sera from a survey of 6,026 people were tested for hepatitis B surface antigen by using radioimmunoassay and counterelectrophoresis. Forty-eight sera (0.79%) were positive by counterelectrophoresis and 152 sera (2.52%) were positive by radioimmunoassay, using the most liberal of the recommended criteria for positivity (i.e., counts 3 standard deviations above the mean). Absorption tests performed on the 152 radioimmunoassay-positive sera showed that 10 (6.6%) were false-positive reactions to guinea pig protein, 74 (48.6%) were due to false-positive reaction(s) with other protein(s) in the test system, and 68 (44.8%) were true positives. There was a strong correlation between the degree of elevation of radioactive counts and the proportions of sera that were true positives; all 49 sera with counts >50 standard deviation units above the mean were true positives, but only 19 (18.4%) of the 103 sera with counts <50 standard deviation units were true positives. A few sera with high counts required absorption with type-specific (type D) antisera. The following conclusions were reached from this study: (i) absorption tests should be run on all radioimmunoassay-positive, counterelectrophoresis-negative sera; (ii) most (about 90%) false positives are not due to anti-guinea-pig protein reactions; and (iii) radioimmunoassay, in combination with absorption tests, yields a modest increase (about 35%) in detection of true positives over use of counterelectrophoresis alone.     

Prediction of Clinical Outcomes in Hepatitis B E Antigen Negative Chronic Hepatitis B Patients with Elevated Hepatitis B Virus DNA Levels

Objectives

We investigated whether long-term clinical outcomes such as disease progression or inactive hepatitis B virus (HBV) carrier state can be predicted by baseline factors in hepatitis B e antigen (HBeAg)-negative HBV infected patients with an elevated viral load.

Methods

A retrospective cohort of 527 HBeAg-negative chronic HBV infected patients with an elevated viral load (HBV DNA ≥ 2,000 IU/ml) was assessed for disease progression defined by the development of hepatocellular carcinoma (HCC) or cirrhotic complication, as well as becoming an inactive carrier.

Results

During a median 3.6 years of follow-up, disease progression was detected in 46 patients (40 with HCC, 6 with cirrhotic complication), and 31 of 309 non-cirrhotic patients became inactive carriers. Older age, male gender, cirrhosis, high HBV DNA levels at baseline, and short antiviral therapy duration were independent risk factors for HCC. Low HBV DNA and quantitative hepatitis B surface antigen (qHBsAg) levels were independent predictors for becoming inactive carriers in patients without cirrhosis. In non-cirrhotic patients with both low qHBsAg and HBV DNA levels, the 5-year cumulative incidence of an inactive carrier was 39.8%, while that of disease progression was 1.6%.

Conclusion

HBeAg negative patients without cirrhosis can be closely monitored for becoming an inactive carrier when both HBV DNA and qHBsAg levels are low, as the risk of disease progression is low while incidence of an inactive carrier is high.     

Prevalence of Hepatitis B Antigen and Antibody in Prostitutes

We investigated the prevalence of hepatitis B antigen (HBAg) and antibody (HBAb) in 293 prostitutes and in 379 pregnant women of similar age and of low socioeconomic level, who served as controls. HBAg was found in 4·4% of prostitutes and 3·4% of controls. The prevalence of HBAb was significantly higher (P <0·001) in prostitutes (56·7%) than in controls (24·5%). The prevalence of HBAb was clearly age-dependent in both groups. Evidence of hepatitis B virus infection significantly increased with the number of years in prostitution. The evidence of increased infection rates among prostitutes and their distribution support the hypothesis that hepatitis B infection is sexually transmitted.