Anti-HBcAg monoclonal antibodies from mouse ascites were purified by using immobilized metal ion affinity chromatography. We optimized the conditions of sample loading and elution. The results showed that when the pH stepwise elution was used, the best solution for sample loading was 20 mmol/L phosphate buffer containing 0.5 mol/L sodium chloride at pH 8.0 and the mAb was eluted at pH 5.0. The purity of obtained mAb was more than 85% and recovery reached 80%. When the adsorbed proteins were eluted by using gradient elution of an imidazole, the best solution for loading condition was 20 mmolL phosphate buffer containing 5 mmol/L imidazole at pH 8.0. The purity and recovery of antibody were up to 95%.
Animals ; Antibodies, Monoclonal ; isolation & purification ; Chromatography, Affinity ; methods ; Chromatography, Ion Exchange ; methods ; Hepatitis B Core Antigens ; immunology ; Hydrogen-Ion Concentration ; Imidazoles ; chemistry ; Metals ; chemistry ; Mice

BACKGROUND/AIMS: Alcohol and the hepatitis B virus (HBV) exert synergistic effects in hepatocelluar carcinogenesis. We aimed to elucidate the clinical significance of the antibody to hepatitis B core antigen (anti-HBc) and occult HBV infection on the development of hepatocellular carcinoma (HCC) in patients with alcoholic liver cirrhosis (LC). METHODS: Patients with alcoholic LC alone (n=193) or combined with HCC (n=36), who did not have HBsAg or antibody to hepatitis C virus were enrolled. Clinical data and laboratory data including anti-HBc were investigated at enrollment. The polymerase chain reaction was applied to HBV DNA using sera of patients with HCC or LC after age and sex matching. RESULTS: Patients with HCC were older (60+/-11 years vs. 53+/-10 years, mean+/-SD, P<0.001), more likely to be male (100% vs. 89%, P=0.03), and had a higher positive rate of anti-HBc (91.2% vs. 77.3%, P=0.067), and a higher alcohol intake (739+/-448 kg vs. 603+/-409 kg, P=0.076) than those with LC. Age was the only significant risk factor for HCC revealed by multiple logistic regression analysis (odds ratio, 1.056; P=0.003). The positive rate of anti-HBc and alcohol intake did not differ in age- and sex-matched subjects between the LC (n=32) and HCC (n=31) groups. However, the detection rate of serum HBV DNA was higher in the HCC group (48.4%) than in the LC group (0%, P<0.001). CONCLUSIONS: Anti-HBc positivity is not a risk factor for HCC. However, occult HBV infection may be a risk factor for HCC in patients with alcoholic LC.
Adult ; Aged ; Antibodies, Viral/blood ; Carcinoma, Hepatocellular/diagnosis/epidemiology/*etiology ; DNA, Viral/analysis ; Female ; Hepatitis B/*complications/diagnosis ; Hepatitis B Core Antigens/*immunology ; Hepatitis B Surface Antigens/immunology ; Hepatitis B virus/genetics/immunology/isolation & purification ; Hepatitis C/complications/diagnosis ; Humans ; Liver Cirrhosis, Alcoholic/*complications/diagnosis/epidemiology ; Liver Neoplasms/diagnosis/epidemiology/*etiology ; Male ; Middle Aged ; Risk Factors

BACKGROUNDS/AIMS: Negative hepatitis B core antigen (HBcAg) staining in hepatocytes is indicative of viral replication by an active immune response. HBcAg is expressed mainly in the cytoplasm in patients with active hepatitis and hepatocyte regeneration, and mainly in the nuclei of hepatocytes in patients with minimal liver injury in the absence of hepatocyte regeneration. The aim of this study was to elucidate whether the existence and expression pattern of HBcAg predicts the response to antiviral treatment. METHODS: The study involved 58 patients with biopsy-proven chronic hepatitis B who were treated with lamivudine. Hepatitis B e antigen (HBeAg), antibody to HBeAg, hepatitis B virus DNA, and alanine aminotransferase in serum were recorded every 3 months. The inflammation grade and the fibrosis stage of chronic hepatitis were scored from 0 to 4 according to lobular inflammation, portal inflammation, periportal inflammation, and fibrosis. RESULTS: The 58 patients included 49(84%) HBcAg-positive patients, with HBcAg staining confined to the cytoplasm in 15(31%) and in both cytoplasm and nuclei in 34(69%). The grade of lobular inflammation and the total histology score were significantly higher in patients with cytoplasmic expression of HBcAg than in HBcAg-negative patients (lobular inflammation: 2.9 vs 2.1, P=0.02; total histology score: 12.2 vs 10.3, P=0.04). The virologic responses at 3, 6, 9, and 12 months differed significantly between the cytoplasmic and mixed expression groups (P<0.01). CONCLUSIONS: The expression pattern of HBcAg (including its possible absence) before initial therapy appears to predict the response to antiviral treatment.
Adult ; Antiviral Agents/*therapeutic use ; DNA, Viral/blood ; Female ; Hepatitis B Core Antigens/*analysis/metabolism ; Hepatitis B e Antigens/metabolism ; Hepatitis B virus/isolation & purification ; Hepatitis B, Chronic/*drug therapy/virology ; Humans ; Lamivudine/*therapeutic use ; Male ; Middle Aged ; Predictive Value of Tests ; Retrospective Studies

<b>OBJECTIVEb>To establish a mismatched polymerase chain reaction restricted fragment length polymorphism (mPCR-RFLP) method for detection of hepatitis B virus (HBV) mutation in precore A1896, and compare with direct sequencing for evaluating its applicability.

<b>METHODSb>According to the principle of mPCR, 194bp gene fragments in HBV precore region was amplified. The products of PCR were digested by Bsu36I and subjected to agarose gel electrophoresis. A method for detecting procore A1896 mutation was established by restricted fragment length polymorphism. Totally 134 sera were analyzed by both mPCR-RFLP and direct sequencing methods. Two sera which were identified having mixed infection with precore wild and mutant strains by mPCR-RFLP also were analyzed by cloning and sequencing.

<b>RESULTSb>From 134 sera, 117 could be analyzed for HBV precore 1896 situation by mPCR-RFLP method, 109 could be analyzed by sequencing. In 101 sera which could be analyzed by the two methods, 54 were mutant strains and 47 were wild strains. The results of both methods were completely compatible. There was no significant difference in detective rate of HBV precore A1896 mutation between the two methods. The sequences of five clones from one serum which was identified precore mutant by mPCR-RFLP were all A1896 mutant strains. Another serum was identified as mixed infection by mPCR-RFLP, one clone was A1896 mutant strain and four were G1896 wild strains. The results of mPCR-RFLP were verified by cloning.

<b>CONCLUSIONb>Compared with sequencing, the mPCR-RFLP method is simple, accurate and can be used in large-scale surveys and clinical research.

Adult ; Aged ; DNA, Viral ; blood ; genetics ; isolation & purification ; Female ; Genetic Heterogeneity ; Hepatitis B ; blood ; virology ; Hepatitis B Core Antigens ; genetics ; Hepatitis B virus ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Young Adult

<b>OBJECTIVEb>To elucidate the relationship between HBV core promoter mutation and clinical features as well as its effects on serum e system and viral replication.

<b>METHODSb>Semi-nested mutation specific PCR (msPCR) was employed for detecting core promoter mutation at nt 1 762-1 764 in 97 patients with HBV infection.

<b>RESULTSb>The msPCR method was demonstrated to be specific and reliable for the mutation detection by sequencing the PCR products. The detection ratio of the mutation in patients with acute hepatitis, mild, moderate and severe chronic hepatitis and liver cirrhosis was 2/5, 7/43, 10/31, 1/3 and 7/15, respectively. The detection rate of the mutation in liver cirrhosis was significantly higher than that in light chronic hepatitis (P < 0.025). In 92 patients with chronic HBV infection, HBeAg positive rate in wild (25/92), mutant (42/92) and mixed (25/92) strain infection was 80.0%, 56.0% and 64.3%, HBV DNA level was (4.4 +/- 8.5) x 10(8), (1.1 +/- 1.6) x 10(9) and (1.4 +/- 1.8) x 10(9) copies/ml, the rate of abnormal ALT was 44.0%, 52.0% and 42.6%; ALT level was (58.6 +/- 79.0), (57.1 +/- 75.2) and (62.6 +/- 90.3) IU/L, respectively (P > 0.05).

<b>CONCLUSIONSb>The msPCR method for detecting core promoter mutation at nt 1 762-1 764 is specific and reliable. Core promoter mutation is associated with the severity of liver disease, but neither related to the status of e system in serum nor to the virus replication.

Adolescent ; Adult ; Aged ; Child ; DNA, Viral ; blood ; genetics ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis B ; blood ; pathology ; virology ; Hepatitis B Core Antigens ; blood ; genetics ; Hepatitis B virus ; genetics ; isolation & purification ; physiology ; Host-Pathogen Interactions ; Humans ; Male ; Middle Aged ; Mutation ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Virus Replication ; Young Adult

Adult ; Female ; Hepatitis B Core Antigens ; analysis ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B virus ; isolation & purification ; Hepatitis B, Chronic ; virology ; Humans ; Ovary ; virology

BACKGROUND/AIMS: Although the viral load is correlated with HBcAg, liver injury was not correlated to viral load in HBeAg positive patient. We aimed to study the inter-relationship of clinical parameters such as the level of HBV-DNA, the level of aminotransferase, intrahepatic expression of HBcAg and severity of histological liver damage in the young male chronic HBV carriers according to HBeAg status. METHODS: The study group included 85 young male patients (mean age: 19.8) with biopsy-proven chronic hepatitis B (HBeAg-positive group: n=60, HBeAg-negative group: n=25). RESUTLS: Serum levels of HBV-DNA and the expression of intrahepatic HBcAg in the HBeAg-positive group were significantly higher than in the HBeAg-negative (p<0.001), but fibrosis score was lower (p<0.01). Serum levels of HBV-DNA positively correlated with lobular activity, portal/periportal activity, biochemical activities in the HBeAg-negative group but negatively correlated in the HBeAg-positive group. There were no significant differences in histological activity according to the pattern of expression of intrahepatic HBcAg in both groups. The lobular activity correlated positively with biochemical activity in both groups, and portal/periportal activity correlated with biochemical activity only in the HBeAg-positive group. CONCLUSIONS: There are close correlations among liver injury, intrahepatic expression of HBcAg, and detectable HBV-DNA in the young male chronic HBV carriers with HBeAg-negativity, but in the HBeAg-positive group, the correlations are diversified.
Adolescent ; Adult ; DNA, Viral/*analysis ; English Abstract ; Hepatitis B Core Antigens/*analysis ; Hepatitis B virus/genetics/*isolation & purification ; Hepatitis B, Chronic/pathology/*virology ; Humans ; Liver/*pathology/virology ; Male ; Viral Load

<b>OBJECTIVEb>To study the characteristics of full-length Hepatitis B Virus (HBV) genomes isolated from patients with hepatocellular carcinoma (HCC).

<b>METHODSb>The full-length HBV genomes from the serum of HBsAg positive HCC patients were amplified by PCR, and then sequenced and analyzed its structure.

<b>RESULTSb>Twenty-two full-length HBV DNAs were obtained from different patients of HCC. Phylogenetic analysis revealed that all HBV strains could be categorized into genotype B or C and serotype adr or adw2. Structural analysis showed that HBV obtained shared meaningful consensus mutations in B/T cell epitopes of surface and core antigens, transactivating domain of X protein and enhancer II/core promoter regions as compared to standard strains.

<b>CONCLUSIONb>Genotype and gene mutation of HBV may be closely correlated with the carcinogenesis of HBV-related hepatocellular carcinoma.

Amino Acids ; genetics ; Carcinoma, Hepatocellular ; virology ; Cloning, Molecular ; DNA, Viral ; genetics ; Genome, Viral ; Genotype ; Hepatitis B Core Antigens ; genetics ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B virus ; genetics ; immunology ; isolation & purification ; Humans ; Liver Neoplasms ; virology ; Mutation ; Phylogeny ; Promoter Regions, Genetic ; Trans-Activators ; genetics

Biomarkers ; blood ; DNA, Viral ; blood ; Female ; Hepatitis B ; genetics ; immunology ; prevention & control ; Hepatitis B Antibodies ; biosynthesis ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; immunology ; Hepatitis B e Antigens ; blood ; immunology ; Hepatitis B virus ; isolation & purification ; Humans ; Male ; Vaccination

<b>OBJECTIVEb>To investigate whether the hepatitis B virus (HBV) has quasispecies character by studying nucleotide sequence polymorphism and mutation features of HBV PreC/C gene region, and preliminaryly explore the heterogeneity of HBV quasispecies.

<b>METHODSb>The serum sample was obtained from a patient with chronic hepatitis B, and the whole HBV PreC/C gene region was amplified by PCR and cloned. Thirty-four clones that contained HBV PreC/C gene fragments were sequenced.

<b>RESULTSb>There were 28 kinds of different nucleotide sequences in 34 clones, and the nucleotide sequences diversity ranged from 0.2% to 2.1%. The mutation points were almost distributed in the whole region, but there wasn't mutation at PreC region nt.1 896 point in all sequences.

<b>CONCLUSIONb>Hepatitis B virus has complex quasispecies character in the patients with chronic hepatitis B.

Adult ; Hepatitis B Core Antigens ; genetics ; immunology ; Hepatitis B virus ; classification ; genetics ; isolation & purification ; Hepatitis B, Chronic ; virology ; Humans ; Male ; Mutation ; Promoter Regions, Genetic ; genetics ; Sequence Analysis, DNA