In order to produce recombinant human anti-HBsAg Fab antibody in Pichia pastoris, the recombinant yeast was fermented using fed-batch system in a 30 L bioreactor. The fermentation temperature was 30 degrees C, the pH was 5.0 approximately 5.3, and the DO was 20% approximately 30%. The recombinant Fab antibody was purified from crude culture supernatant by ion exchange and analyzed by SDS-PAGE and western blot and ELISA. When the absorbance (OD600) of broth reach 300 at the end of fed-batch phase, the induced phase was initiated. The results showed that recombinant human anti-HBsAg Fab antibody was high-level expressed in recombinant Pichia pastoris using a fed-batch fermentation system. Both chains of the Fab were successfully expressed upon methanol induction. After 192 h of induction, the expression level of recombinant Fab (soluble) reached 412 mg/L. The recombinant Fab antibody was purified effectively by ion-exchange chromatography from the fermentation supernatant to a purity of 95%. And the affinity activities of the purified recombinant Fab antibdy and fermentation supernatant were detected, and both of them showed high affinity activities. The results demonstrated that recombinant human anti-HBsAg Fab antibody could be high level produced by fed-batch fermentations in Pichia pastoris. Which can be efficiently used in industrial production.
Fermentation ; Hepatitis B Antibodies ; biosynthesis ; isolation & purification ; Hepatitis B Surface Antigens ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; isolation & purification ; Pichia ; genetics ; Recombinant Proteins ; biosynthesis ; isolation & purification

Thirty turtles (15 Clemys mutica and 15 Geoclemys reevesii) which were inoculated with human sera those were positive for hepatitis B surface antigen (HBsAg) and hepatitis B "e" antigen (HBeAg) were found to be infected with hepatitis B virus (HBV). The levels of HBV infection markers, such as HBsAg and antibody to HBsAg (anti-HBsAg), were retinely monitored in the turtles' serum for 46 weeks. Within two weeks of the inoculation, 42% of the turtles tested were positive for HBsAg, and their reciprocal titers as measured by reverse passive hemagglutination (RPHA) and enzyme linked immunoabsorbance assay (ELISA) ranged from 16 to 96. Within 20 weeks, the remaining turtles tested HBsAg positive, as confirmed by ELISA. At 20 weeks, all but one of the turtles exhibited changes in HBV blood marker from HBsAg to anti-HBs; the one exception was positive for both HBsAg and anti-HBs. At the 47th week, 7 animals were killed and their organs were examined for HBV infected cells utilizing an immunofluorescent technique. Numerous fluorescent cells which reacted with human anti-HBs nad anti-HBc were observed in the following organs: pancreas, liver, kidney, and brain. Histopathologically, edematous changes in hepatocytes and minor cellular infiltration attributed to an inflammatory response were noted. Liver and kidney cells from the infected animals were cultured, and HBV antigen positive cells for HBsAg and HBcAg were detected in the cultures. Throughout the experiment, HBsAg was detected in the supernatant by ELISA. Virus particles which were indistinguishable from Dane particles were seen in the cytoplasmic vacuoles of the cultured cells by electron microscopy. Finally, the presence of HBV DNA was established by molecular hybridization techniques in the culture supernatants of kidney cells from the infected turtles.
Animal ; Hepatitis B/microbiology/transmission/*veterinary ; Hepatitis B Antibodies/isolation and purification ; Hepatitis B Core Antigens/isolation and purification ; Hepatitis B Surface Antigens/isolation and purification ; Human ; Kidney/microbiology ; Liver/microbiology/pathology ; Turtles/*microbiology

<b>OBJECTIVEb>To see the HBV DNA detection instance in the HBsAg negative people and to study the serological method detection strategy for detecting hepatitis B virus large surface protein (HBLP) to filtrate the occult HBV infection.

<b>METHODSb>The HBsAg negative serum samples were divided into HBsAb negative and positive two species according to the hepatitis B virus markers (HBVM) in daily work excepting the special HBVM modes. Total 2000 stochastic serum samples with 1000 HBsAb negative results and 1000 HBsAb positive results were collected from the copy tubes to detect HBVM with national ELISA reagent kits and put them -20 degrees C frostily. Mixed samples (8 x 30 microl) were analyzed with fluorescence quantitative PCR (FQ-PCR) and filtrated the individual positive samples. The filtrated samples were doubly tested again with American MONOLISA HBsAg ULTRA reagents.

<b>RESULTSb>No HBV DNA positive results were found out from the 1000 HBsAb positive samples and 19 cases HBV DNA positive results were found out from the 1000 HBsAb negative samples. On these 19 samples, the HBsAg results from the American MONOLISA HBsAg ULTRA reagents were all positive and the HBLP results were all positive, too. The 19 HBV DNA quantitative results were divided into 2 cases more than 500 copies/ml, 3 cases between 400-500 copies/ ml, 3 cases between 300-400 copies/ml, 7 cases between 200-300 copies/ml and 4 cases between 100-200 copies/ml.

<b>CONCLUSIONb>The leaked samples tested HBsAg with national reagents are mostly from the HBsAb negative people. HBLP results may be positive on these samples and detecting HBLP marker is propitious to filtrate the occult HBV infection. This study provided a kind of serological reference for actively searching for the detecting strategy in occult HBV infection field.

Antibody Specificity ; Hepatitis B ; diagnosis ; Hepatitis B Antibodies ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B virus ; isolation & purification ; Humans ; Laboratories ; Polymerase Chain Reaction

<b>OBJECTIVEb>Through detecting the standard preparation with series of concentration to indirectly calculate the anti-HBs concentration of the serum samples, a suitable anti-HBs quantitative method for our laboratory was found after comparing the two kinds of methods.

<b>METHODSb>Detecting the anti-HBs standard preparation with series of concentration by RIA method, standard curvilinear equations were obtained by the means of fitting the detected result and the corresponding concentration by log-log model and exponential curve model respectively. Then the fitting efficiency of two curves was compared. By calculating the concentrations of the reference using two standard curvilinear equations, we can compare the accuracy of two quantitative methods.

<b>RESULTb>The error mean square of the exponential curve model is low as 1.2971 and the determinate coefficient is close to 1 with the value of 0.9904. The average concentrations (n=6) of the detected reference calculated by two curvilinear equation with the actual concentration of 30.0 mIU/mL are (32.28 +/- 1.06) and (31.91 +/- 1.06) mIU/ mL respectively. The concentration calculated by exponential curve model is only 6.37% higher than the actual concentration.

<b>CONCLUSIONb>Fitting by exponential curve model is more practical to estimate the actual concentration of the serum samples those will be detected. It can be used as an optimal quantitative method to detect anti-HBs concentration.

Hepatitis B ; blood ; immunology ; virology ; Hepatitis B Antibodies ; blood ; immunology ; Hepatitis B Surface Antigens ; blood ; immunology ; Hepatitis B virus ; immunology ; isolation & purification ; Humans ; Male ; Radioimmunoassay ; methods

<b>OBJECTIVEb>To analyze the characteristic of T cell response to specific antigen proteins in patients with hepatitis B virus infection.

<b>METHODSb>76 cases were recruited, including four groups, acute hepatitis B (AHB), active phase of chronic hepatitis B (CHB), inactive HBV carriers (AsC) and past HBV infection. T cell responses stimulated by 3 antigen specific proteins of HBV were detected using enzyme linked immunospot (ELISPOT) assay.

<b>RESULTSb>(1) There were no significant difference in frequencies to HBsAg, HBcAg and HBeAg in AHB and CHB. The frequencies to HBsAg and HBcAg in AsC were lower than that to HBeAg, and the frequencies to HBsAg in group of past HBV infection were significantly lower than that to HBcAg and HBeAg. (2) The frequencies to HBsAg in AHB and CHB both were higher than in group of past HBV infection. The frequencies to HBcAg of AHB, CHB and AsC were higher than that of group of past HBV infection. (3) There were no significant difference in magnitude to HBsAg, HBcAg and HBeAg in AHB and AsC. In CHB, the magnitude to HBsAg was lower than that to HBcAg. The magnitude of in group of past HBV infection were HBcAg > HBeAg > HBsAg. (4) In four groups, the sequence of the magnitude to HBsAg from high to low was AHB, CHB, group of past HBV infection and AsC. The magnitude to HBcAg in of AsC was lower than other three groups. As to the magnitude to HBeAg, the difference was no significant between any two groups except between AHB and CHB.

<b>CONCLUSIONSb>The T cell responses in group of AsC to HBeAg were the highest, while the T cell responses to HBcAg were the highest in group of other groups.

Hepatitis B ; immunology ; virology ; Hepatitis B Antibodies ; immunology ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B e Antigens ; immunology ; Hepatitis B virus ; immunology ; isolation & purification ; Humans ; T-Lymphocytes ; immunology

<b>OBJECTIVEb>To know genotypes and serotypes of hepatitis B virus (HBV) detected from hepatitis B infected people in Yunnan Province.

<b>METHODSb>Serum samples were collected from HBsAg carriers detected from people who had a physical examination at Yunnan Provincial Center for Disease Control and Prevention. The S genes of HBV were amplified by nested PCR and the PCR products were sequenced. The viral genotype was identified by phylogenetic analysis. 27 reference sequences corresponding to HBV genotype A to I were obtained from GenBank. According to the amino acid sequences deduced from the nucleotide sequences of S gene, the dominant serotype of HBV detected from these people were confirmed.

<b>RESULTSb>39 HBsAg positive serum samples were detected from 2216 people who had a physical examination. The results shows that 76.9% were C genotype; 15.4% were B genotype; 5.1% were D genotype; 2.5% were I genotype. Three serotypes were found. The rates of adw2, adrq+ and ayr serotypes are 71.8%, 17.9% and 10.3% respectively. All of adw2 subtype specimens are C genotype. Among the serum specimens in which both HBsAg and HBeAg are positive, 75% were C genotype and adw2 subtype.

<b>CONCLUSIONb>It is determined that the main genotype and subtype of HBV prevailed in Yunnan province is C genotype and adw2 subtype.

Antibodies, Viral ; immunology ; China ; Female ; Hepatitis B ; virology ; Hepatitis B Surface Antigens ; genetics ; immunology ; Hepatitis B virus ; classification ; genetics ; immunology ; isolation & purification ; Humans ; Male ; Physical Examination ; Population Surveillance

DNA, Viral ; blood ; Female ; Hepatitis B ; immunology ; virology ; Hepatitis B Antibodies ; blood ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Male ; Polymerase Chain Reaction ; methods

<b>BACKGROUNDb>To establish a new assay for detecting the quantity of HBV DNA with PCR and enzyme-linked immunosorbent assay(ELISA).

<b>METHODSb>The products of PCR using primers pre-labeled with biotin were hybridized with the capture probes that were immobilized on the microtiter strips and then bond with Sav-Ap. The quantity of DNA was detected by measuring the yellow color at 405 nm wave length.

<b>RESULTSb>Totally 125 sera from patients with hepatitis B were tested for HBV DNA by this method,the sera were also tested for HBV immunological markers by solid phase radio immuno-assay (SPRIA). The HBV positive rate with PCR-ELISA was 64.9% (24/37) in samples which were positive for HBsAg, HBeAg and HBcAb; and 34.2% (13/38) in sera which were positive for HBsAg, HBeAb and HBcAb; in sera positive for HBsAg and HBcAb or only HBcAb, the positive rate was 6.7% (1/15) and 5.9% (2/34) respectively.

<b>CONCLUSIONSb>The PCR ELISA assay is simple and suitable for clinical laboratory in quantitative determination of HBV DNA.

DNA Probes ; DNA, Viral ; analysis ; biosynthesis ; genetics ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B ; virology ; Hepatitis B Antibodies ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction ; Sensitivity and Specificity

<b>OBJECTIVEb>This study was designed to explore the incidence rate of occult HBV infection in patients with anti-HBc positive alone and analyze the possible reasons of occult infection.

<b>METHODSb>Sera of 183 patients carrying anti-HBc alone(A < or = 0.1) were collected and real-time PCR was used to select samples with HBV DNA positive. HBV pre-S/S amplification products were obtained by PCR, and clonal sequencing were then used for these samples with HBV DNA positive.

<b>RESULTSb>DNA quantitative results of three samples were greater than 10(3) copies/ml in 183 samples, with a fraction of 1.6%. Pre-S/S sequencing results of two samples from these three samples were obtained. Point mutations within "a" determinant with Q129R/P mutations and co-existence of the mutant type and wild type were found in the two samples.

<b>CONCLUSIONSb>Occult HBV infection existed in samples with anti-HBc alone. Factors contributing to the loss of HBsAg detection by immunoassays include S gene mutations and low levels of circulating antigen which are below the assay limit of detection. Occult HBV infection not only can lead to a false clinical diagnosis, but also can result in hematological pollution due to such occult infection of blood donors.

Base Sequence ; Blood Donors ; DNA, Viral ; analysis ; Genotype ; Hepatitis B ; diagnosis ; immunology ; Hepatitis B Antibodies ; Hepatitis B Core Antigens ; isolation & purification ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B virus ; genetics ; immunology ; Humans ; Polymerase Chain Reaction ; Protein Precursors ; genetics

In order to find out the distribution of Genotype of those people infected with HBV (hepatitis B virus) from north Guangxi and the relationship between different immune status of HBV infected people and their genotypes, the HBV infected people are classified into three types according to immune tolerance, immune clearance ( response) and immune incompetence (residues). 150 cases from each type, a total of 450 cases are chosen to be tested with real time fluorescence quantitative PCR assay for detection of HBV infection in three kinds of different immune state of the HBV genotype. In the 450 cases, 323 cases belong to type B, 94 cases belong to type B, 23 cases belong to mixed type B+C and 10 cases belong to none B and none C type. Type B are the majority in all the three HBV immune status, made up to 70%, 78%, 67.33% of each type. The different immune state genotype proportion difference don't have statistical significance; immune state and genotypic correlation isn't statistically significant; type B HBV-DNA load is higher than that of type C, groups of persons aged 30 years or older with type C are significantly higher than that of < 30 years of age, the difference was statistically significant; among the genotypes of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil) positive rate showed no significant difference between male and female; there was no significant difference in genotype distribution. The results show that, in North Guangxi HBV genotypes B, C accounts for the proportion, a small amount of B+C hybrid, occasionally fails to type HBV infection; among immune tolerance, immune clearance (response) and immune incompetence (residues) type B are in majority in these three kinds of immune state, chronic HBV infection immunity with the HBV genotype correlations were not statistically significant.
Adult ; Aged ; China ; Female ; Genotype ; Hepatitis Antibodies ; immunology ; Hepatitis B ; immunology ; virology ; Hepatitis B virus ; classification ; genetics ; immunology ; isolation & purification ; Humans ; Male ; Middle Aged ; Young Adult