Positive clones from Pichia pastoris strain GS115-47-4 and KM71-47-1 were grown in 1200 ml BMGY/BMMY medium into 2 liters fermentor and 50 ml innoculum with high density was added, temperature was 30oC, agitation speed was 300 rpm and pressure was 0.5 kg/cm2. The biomass was harvested after 72 h of induction in case of GS115-47-4 strain and 48 h for KM71-47-1 strain. The density was 3,9 x 109 cells/ml. After cell lysis, the total HBsAg contents were obtained approximately 30 mg in both strains. 2 consecutive isopycnic KBr gradient ultracentifugations following one rate-zonal sucrose were performed.
hepatitis B vaccines ; isolation & purification ; Hepatitis B Surface Antigens

China ; epidemiology ; Hepatitis B ; classification ; epidemiology ; virology ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Mutation

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

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

<b>OBJECTIVEb>To investigate the viral contamination of invasive medical instruments in dentistry and to provide health administrative institutions with surveillance data.

<b>METHODSb>Sterilized samples were randomly collected from the department of dentistry to detect HBV-DNA, HCV-RNA, HIV-RNA and HBsAg.

<b>RESULTSb>Of the invasive medical instruments that were sterilized with 2% glutaraldehyde, one of the samples was positive for HBV-DNA, and another sample was positive for HBsAg.

<b>CONCLUSIONb>Though massive virus contamination of invasive medical instruments in dentistry has been reduced to a low level, the occurrence of contamination still remains.

DNA, Viral ; analysis ; Dental Instruments ; virology ; Equipment Contamination ; HIV ; isolation & purification ; Hepacivirus ; isolation & purification ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B virus ; isolation & purification ; Humans ; RNA, Viral ; analysis

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 establish enzyme-linked immunosorbent assay (ELISA) for detection of hepatitis B virus large surface protein(HBV-LP) in serum.

<b>METHODSb>A sandwich reaction was preformed with horseradish peroxidase labeled monoclonal antibody of HBV-LP as the catalytic enzyme. Several reactions liquid's concentration and reaction conditions were optimized. The method was evaluated in all aspects such as sensitivity, specificity, stability and so on.

<b>RESULTSb>The detection limit was 5 ng/ml. Interassay and intra-assay RSD were both less than 10%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0.98 and RSD lower than 10%.

<b>CONCLUSIONb>Established ELISA for determination of serum HBV-LP has high sensitivity and repeatability. Enzyme-linked immunosorbent assay;

Enzyme-Linked Immunosorbent Assay ; methods ; Hepatitis B ; blood ; diagnosis ; virology ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; isolation & purification ; metabolism ; Humans