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

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

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

Using of two-step integrating technology, transducted the H and L chain gene of humanized Fab fragment of anti-HB-sAg antibody into the genome of methylotropic yeast P. pastoris. Constructed a engineering yeast to produce humanized Fab fragment of the anti-HBsAg antibody. The Fab fragment was efficiently secreted into the medium at a concentration of 50-80 mg/L. The Fab fragment was purified from culturing supernatant of the recombinant yeas by affinity chromatography. The ELISA analysis showed the high affinity of the expressed humanized Fab fragment to the HBsAg.
Chromatography, Affinity ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B Antibodies ; biosynthesis ; genetics ; isolation & purification ; Hepatitis B Surface Antigens ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; genetics ; isolation & purification ; Pichia ; genetics ; Recombinant Proteins ; biosynthesis ; isolation & purification

<b>OBJECTIVEb>To develop an amplified luminescent proximity homogeneous immunoassay (AlphaLISA) kit for the detection of human hepatitis B virus e antibody (HBeAb).

<b>METHODSb>The neutralizing and competitive inhibition method was used to develop the AlphaLISA kit for detection of serum HBeAb.

<b>RESULTSb>The working range of the kit was 0.003-16 NCU/ml with a sensitivity up to 0.003 NCU/ml. The intra- and inter-assay coefficient of variation was 5.3% and 6.8%, respectively. The kit showed no cross-reaction with HBcAb, and comparison of the detection results with those of a commercially available Elecsys HBeAb kit (Roche) for 136 samples showed a correlation coefficient of 0.961.

<b>CONCLUSIONb>The AlphaLISA kit for HBeAb detection meets the clinical requirements for detection HBeAb in human serum.

Equipment Design ; Hepatitis B Antibodies ; blood ; isolation & purification ; Humans ; Immunoassay ; instrumentation ; Luminescent Measurements ; instrumentation ; Reagent Kits, Diagnostic

<b>OBJECTIVEb>To analyze the status of hepatitis B virus (HBV) infection in non-Hodgkin lymphoma (NHL) patients.

<b>METHODSb>The serum HBV markers in NHL patients were detected by enzyme-linked immunosorbent assay (ELISA). The infection rate of HBV in NHL patients was compared with that in nationwide general population.

<b>RESULTSb>The positive rates of HBsAg, anti-HBs and anti-HBc in 405 cases of NHL were 11.6%, 39.8% and 47.9%, respectively, which were statistically different from those in general population (P < 0.01). The positive rates of HBsAg, anti-HBs and anti-HBc in B-cell NHL and T-cell NHL were 13.3% vs 7.1% (P = 0.083), 40.6% vs 37.5% (P = 0.567), 53.2% vs 33.9% (P = 0. 001), respectively. The HBV DNA positive rate was 23.7% in 93 cases of NHL, and was 50.0% in 38 cases of HBsAg-positive NHL while 5.5% in 55 cases of HBsAg-negative but HBcAb-positive NHL.

<b>CONCLUSIONSb>The infection rate of HBV in NHL patients is higher than that in general population, in which occult hepatitis B virus infection can not be ignored. The positive rate of anti-HBc in B-cell NHL is significantly higher than that in T-cell NHL. For NHL patients infected with HBV, prophylactic anti-HBV therapy to prevent viral reactivation should be given before the anti-cancer treatment. Further study in the relationship between HBV and NHL should be carried out in the future.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; DNA, Viral ; blood ; Female ; Hepatitis B ; epidemiology ; Hepatitis B Antibodies ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; isolation & purification ; Humans ; Lymphoma, Non-Hodgkin ; virology ; Male ; Middle Aged ; Retrospective Studies ; Young Adult

<b>OBJECTIVEb>To optimize the adsorption condition of cation-exchange chromatographic media Streamline SP for separation and purification of anti-HBsAg Fab fragment from E. coli.

<b>METHODSb>The adsorption of the target protein for separation and purification by the cation-exchange chromatographic media Streamline SP was tested using test tube method in balanced buffer solution with different pH values and ion concentrations. The adsorption effect was then verified by cation-exchange chromatography using 1-ml Streamline SP prepacked column and 28-ml Streamline SP self-assembly column.

<b>RESULTSb>According to the experiment results of test tube method, the loading buffer with pH of 4.4 and ionic concentration of 100 to 600 mmol/L could achieve optimal target protein adsorption effect by cation-exchange chromatographic media Streamline SP, as verified by cation-exchange chromatography with 1-ml SP prepacked column and 28-ml Streamline SP self-assembly column.

<b>CONCLUSIONb>The optimal condition of cation-exchange chromatography selected by test tube method can be applied for separation and purification of anti-HBsAg Fab fragment from E. coli.

Adsorption ; Cation Exchange Resins ; Chromatography, Ion Exchange ; methods ; Escherichia coli ; genetics ; metabolism ; Hepatitis B Antibodies ; isolation & purification ; metabolism ; Hepatitis B Surface Antigens ; immunology ; Humans ; Immunoglobulin Fab Fragments ; isolation & purification ; metabolism

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