Autoantibodies in Chinese patients with chronic hepatitis B: prevalence and clinical associations.

AIM: To investigate the prevalence of autoantibodies and their associations with clinical features in Chinese patients with chronic hepatitis B (CHB). METHODS: A total of 325 Chinese patients with CHB were enrolled in this retrospective, hospital-based study. Patients with chronic hepatitis C (CHC), autoimmune hepatitis (AIH), or primary biliary cirrhosis (PBC) were included, with healthy donors acting as controls. A panel of autoantibodies that serologically define AIH and PBC was tested by indirect immunofluorescence assay and line immunoassay. The AIH-related autoantibody profile included homogeneous anti-nuclear antibodies (ANA-H), smooth-muscle antibodies, anti-liver kidney microsome type 1, anti-liver cytosolic antigen type 1, and anti-soluble liver antigen/liver pancreas; the PBC-related antibodies were characterized by ANA-nuclear dots/membranous rim-like, anti-mitochondrial antibodies-M2 (AMA-M2), anti-BPO (recombinant antigen targeted by AMA-M2), anti-Sp100, anti-promyelocytic leukemia protein (anti-PML), and anti-gp210. The dichotomization of clustering was used to unequivocally designate the AIH or PBC profiles for each case. Anti-Ro52 antibodies were also tested. RESULTS: The prevalence of any autoantibody in CHB amounted to 58.2%, which was similar to the 66.2% prevalence in CHC, significantly higher than the 6.7% in the healthy controls (P < 0.001), and lower than the 100% found in AIH and PBC (P = 0.004 and P < 0.001, respectively). There were more anti-PML and anti-gp210 antibodies among the CHB patients than the CHC patients (11.1% vs 0%, P = 0.003; 12.6% vs 0%, P < 0.001, respectively). The prevalence and titer of AMA, anti-BPO, anti-PML, and anti-gp210 were higher in PBC than in those with CHB. Among the CHB patients, the prevalence of ANA, especially ANA-H, was significantly lower in patients with compensated and decompensated cirrhosis compared with patients without cirrhosis. Thirty-eight cases of hepatocellular carcinoma (HCC) in CHB showed a significant difference compared with non-HCC patients in the prevalence of anti-PML (0% vs 12.5%, P = 0.013). Dichotomization of the autoantibodies revealed that the PBC profile was more prevalent in patients with CHB than in those with CHC, and that it was strongly correlated with both compensated and decompensated cirrhosis. In contrast, the prevalence of the AIH profile was significantly higher in non-cirrhosis patients with CHB than in those with compensated cirrhosis (18.5% vs 8.2%, P = 0.039). Moreover, the AIH profile was also closely associated with hepatitis B e-antigen positivity. CONCLUSION: ANA-H could be an indicator of early-stage CHB. Dichotomizing the autoantibody profiles revealed that the PBC profile is strongly associated with cirrhosis in CHB.

[Cloning and expressing of tissue inhibitor of metalloproteinases I gene fragment and preparation of monoclonal antibodies against the recombinant protein].

OBJECTIVE: To prepare the monoclonal antibody (mAb) against tissue inhibitor of metalloproteinases I (TIMP-I) fusion protein. METHODS: TIMP-I gene was amplified from fibrotic human liver tissue by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-TIMP-I and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot were used to detect specificity of mAbs. RESULTS: The prokaryotic plasmid expressing the recombinant protein was constructed, and the TIMP-I recombinant protein was expressed and purified. Four hybridoma cell lines that secreted anti-TIMP-I mAbs were obtained. 3 of 4 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with TIMP-I protein. CONCLUSION: The TIMP-I recombinant protein is highly purified and has strong antigenicity. The anti- TIMP-I mAbs were prepared successfully.

[Establishment of a purificatory method for alpha-fetoprotein variant by affinity adsorption].

OBJECTIVE: To establish a purificatory method of alpha-fetoprotein variant (AFP-L3) based on microspincolumn with lens culinaris agglutinin (LCA). METHODS: LCA was isolated by ammonium sulfate precipitation method from lens culinaris. AFP-L3 affinity adsorption microspincolumns which were made from LCA coupled with activated Sepharose 4B were prepared. By adding into the centrifuge column, serum was absorbed and eluted to purify AFP-L3. The results of purified AFP-L3 detection of 10 cases AFP positive sera by electro-chemiluminescence immunoassay were compared with traditional crossed affinity immunoelectrophoresis. RESULTS: 8 of 10 cases AFP-L3 concentration were greater than 5 ng/ml in purified sera. Six cases show positive reaction in affinity immune cross electrophoresis experiment. CONCLUSION: Successfully established purification method of AFP-L3 by affinity absorption based on microspincolumn. The method was more conducive to clinical laboratory applications due to its high sensitive and easy operation.

[The investigantion of comparative experiments for different clinical laboratory instruments].

OBJECTIVE: To estimate the consistency of two VITROS 3600 chemiluminescent analyzers according to the requirement of ISO15189. METHODS: Verification tests were made for precision and accuracy of anti-HCV in two instruments. While 40 serum samples including Anti-HCV negative (10 cases) , positive (10 cases) , and weakly positive (20 cases). and the test results were statistical analised. RESULTS: Two instruments negative and positive control samples intra-batch precision and coefficients of variation were 5% , 4% and 7. 14% , 7. 23% , inter-batch precision and coefficients of variation were 9. 47% , 7. 7% and 8.04%, 7. 6%, are less than requirement CV (15%) by ISO15189. The accuracy of two instrument were 100% , The test results of the control samples showed no significant difference (P < 0. 05). The correlation analysis of the test results of clinical samples R2 =0. 9984, with good consistency. CONCLUSION: Test results of two Vitros 3600 has good consistency and comparability.

[Establishment of a detection method for hepatitis B virus large surface protein (HBV-lP)].

OBJECTIVE: To establish enzyme-linked immunosorbent assay (ELISA) for detection of hepatitis B virus large surface protein(HBV-LP) in serum. METHODS: 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. RESULTS: 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%. CONCLUSION: Established ELISA for determination of serum HBV-LP has high sensitivity and repeatability. Enzyme-linked immunosorbent assay;

[Microplate chemiluminescence enzyme immunoassay for the quantitative analysis of tissue inhibitor of metalloproteinases I].

OBJECTIVE: To establish microplate chemiluminescence enzyme immunoassay (CLEIA) for quantitative analysis of tissue inhibitor of metalloproteinases I (TIMP I) in human serum. METHODS: A sandwich reaction was preformed with horseradish peroxidase(HRP) labeled monoclonal antibody of TIMP I as the catalytic enzyme and the H2O2-luminol as the luminescence reagent. Several physical and chemical parameters were studied and optimized such as immunoreaction conditions, the dilution ratio of TIMP I-HRP, luminescence reaction time and so on. In order to evaluate the method, recovery test, heat stabilization test and comparison test were carried out. RESULTS: The linear range was 0. 2-12 ng/ml with r = 0.996. The detection limit was 0.12 ng/ml. Inter-assay and intra-assay RSD were both less than 10%. The recoveries of three different spiked concentration samples were 100.6%, 96.5% and 106.5%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0. 998 and RSD lower than 6%. The detected results with CLEIA closely corresponded to those with imported ELISA in 60 patients sera with liver fibrosis. CONCLUSION: Established CLEIA for quantity determination of serum TIMP I has high accuracy, sensitivity and repeatability.

[Cloning and expressing of golgi protein73 gene fragment and preparation of monoclonal antibodies against the recombinant protein].

OBJECTIVE: To clone and express human Golgi glycoprotein73 protein, and prepare the monoclonal antibody (mAb) against the protein. METHODS: GP73 gene was amplified from HepG2 cells by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-GP73 and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot was used to detect specificity of mAbs. RESULTS: The prokaryotic plasmid expressing the recombinant protein was constructed, and the GP73 recombinant protein was expressed and purified. Five hybridoma cell lines that secreted anti-GP73 mAbs were obtained. 2 of 5 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with GP73 protein. CONCLUSION: The GP73 recombinant protein is highly purified and has strong antigenicity. The anti-GP73 mAbs were prepared successfully.

[Establishment of a quantitative detection method for Golgi protein73].

OBJECTIVE: To establish enzyme-linked immunosorbent assay (ELISA) for quantitative detection of Golgi protein73 (GP73) in serum. METHODS: A sandwich reaction was preformed with horseradish peroxidase labeled monoclonal antibody of GP73 as the catalytic enzyme. Several reactions liquid's concentration and reaction conditions were optimized. The method was evaluated in all aspects such as linear range, sensitivity, specificity, stability and so on. RESULTS: The linear range was 25-500 ng/ml. The detection limit was 18.5 ng/ml. Inter-assay and intra-assay RSD were both less than 10%. The recoveries of three different spiked concentration samples were 95.3%, 92.6% and 103.7%. 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%. CONCLUSION: Established ELISA for quantity determination of serum GP73 has high accuracy, sensitivity and repeatability.

[Quantitative detection of serum procollagen III N-terminal peptide chemiluminescence enzyme immunoassay].

OBJECTIVE: To establish chemiluminescence enzyme immunoassay (CLEIA) for quantitative detection of procollagen III N-terminal peptide (P III NP) in serum. METHODS: A sandwich reaction was preformed with horseradish peroxidase labeled monoclonal antibody of P III NP as the catalytic enzyme and the luminol as the luminescence reagent. Several reactions liquid's concentration and reaction conditions were optimized. The method was evaluated in all aspects such as linear range, sensitivity, specificity, stability and so on. The CLEIA was compared with imported ELISA kits, by detecting clinical serum. RESULTS: The linear range was 0.8-85 ng/ml. The detection limit was 0.5 ng/ml. Inter-assay and intra-assay RSD were both less than 10%. The recoveries of three different spiked concentration samples were 96.2%, 91.2% and 101.1%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0.99 and RSD lower than 6%. The detected results of clinical sera with CLEIA closely corresponded to those with imported ELISA. CONCLUSION: Established CLEIA for quantity determination of serum P III NP has high accuracy, sensitivity and repeatability.

[Establishment of confirmatory test for suspicious hepatitis B surface antigen positive samples].

OBJECTIVE: Establish a confirmatory test based on ELISA, and use to verify the authenticity of HBsAg weak positive samples, pick and get rid of the false result, and avoid the mistake diagnosis. METHOD: The particles (reagent A) coated by streptavidin and biotinylated HBsAb (reagent B) were mixed in different proportions, then neutralized with serum whose the COI of HBsAg > 20 by ELISA in order to identify the activity of HBsAb in confirmatory reagent. 30 pieces of HBsAg weak positive serum neutralized with the confirmatory reagent, the serum were considered to be positive if rate of decline of HBsAg COI > 50%. The results were compared to Roche confirmatory Kit. RESULT: Confirmatory reagent was able to neutralized with HBsAg. 24 of 30 pieces of HBsAg weak positive samples were judged to be positive, while 6 poeces were negative. The ELISA comfirm method is fully consistent with Roche confirmatory Kit. CONCLUSION: The ELISA confirmatory test for suspicious HBsAg positive samples is a simple, accurate and low cost initial validation method, After further clinical trials, should be widely applied.

[Comparison of two different methods to detect HIV antibodies].

OBJECTIVE: Evaluated the chemiluminescence and enzyme-linked immunosorbent assay (ELISA) to detect HIV antibodies, and compared the results, to provide a reference for the selection and clinical application of HIV screening. METHODS: 3000 cases of our hospital patients were measured by enzyme-linked immunosorbent assay and chemiluminescence immunoassay, using comfirmming experimental results as gold standards. Comparing sensitivity, specificity and other Indicators. RESULTS: In the diagnosis of HIV infection, enzyme-linked immunosorbent assay and chemiluminescence immunoassay had no significant difference. The positive rate of enzyme-linked immunosorbent assay was 0.93%, while the sensitivity and specificity were 89.66%, 99.93%, the positive rate of chemiluminescence immunoassay was 1.03%, while the sensitivity and specificity were 100%, 99.93%, respectively. CONCLUSION: Both methods are suitable for screening of HIV, having high specificity, and chemiluminescence has greater sensitivity than ELISA.

[Establishment of confirmatory test for HBsAb in serum of coexistence of hepatitis B surface antigen and antibodies to HBsAg].

OBJECTIVE: Establish a kind of confirmation method based on ELISA, and use to verify authenticity of HBsAb + in HBsAg + HBsAb + serum, pick and get rid of the false masculine gender the result, and avoid the mistake diagnosis. METHOD: Collect 60 pieces of serum whose thick degree of HBsAg at 1000 COI above tested by ECLIA as confirm serum, mixed the confirm serum of different dilution with HBsAb positive serum to screen and verify best thick degree of HBsAg. Collected 40 pieces of HBsAg + HBsAb + serum, ELISA tested the descend rate of HBsAb COI after neutralized with confirm serum in order to confirm authenticity of HBsAb + in pieces of HBsAg + HBsAb + serum. RESULT: When thick degree of HBsAg is 2000 COI, the performance of neutralization to HBsAb is best. The ELISA confirmatory test is fully consistent with the ECLIA method with true positive of 37 pieces of HBsAg + HBsAb + serum while false-positive of 3 pieces of serum. CONCLUSION: The ELISA confirm method is a simple, accurate and low cost initial validation method.

[Evaluation of the Helicobacter pylori stool antigen test].

OBJECTIVE: To evaluate the clinical value of an enzyme-linked immunosorbent assay by the Helicobacter pylori stool antigen (HpSA) test for the detection of H. pylori infection. METHODS: 328 patients were measured upper gastrointestinal endoscopic examination which as gold standards and HpSA test in the meantime, comparing accuracy, sensitivity, specificity and other Indicators. RESULTS: In the diagnosis of Hp infection, HpSA test had no significant difference comparing with gold standards and had a P value of over 0.5. The sensitivity of HpSA test was 94.6%, while the specificity, veracity, expected positive value, and expected negative value were 96.9%, 96.3%, 89.7% and 98.4%, respectively. CONCLUSION: The H. pylori stool antigen test is a simple, non-invasive method for accurate diagnosis of H. pylori infection.