| Hepatitis B, an infectious disease caused by hepatitis B virus, severely threatens human health. Chronic Hepatitis B virus infection is a globally troubled health problem. There are a number of methods for clinical diagnosis of Hepatitis B. Generally, we make primary diagnosis by HBV serological testing. The HBV serological diagnosis mainly includes:enzyme immunoassay, radioimmunoassay, chemiluminescence, immunofluorescence, and so on, among which the enzyme immunoassay is the most frequently used now. The Hepatitis B antibodies detecting kits usually rely on mammalian IgG. However, the IgG of mammal binds nonspecificly to bacterial Fc receptors, such as staphylococcal protein A or streptococcal protein G, etc.. The most well known one of these anti-mammalian IgG antibodies is the rheumatoid factor (RF) that reacts with the Fc portion of mammalian IgG and the complement system might be activated when a serum sample has been added in an assay. If anti-mammalian IgG antibodies exist in the samples they might cause a false positive reaction. Therefore, it is essential to develop to a new HBV detecting method.This study gained the anti-HBsAg IgYs by immunizing hens with HBsAg. Then, the anti-HBsAg IgYs were linked to HRP by improved sodium iodide oxidation method and at last Double antibody Sandwich ELISA for HBsAg was established.The Romanes laying hens were immunized with 20μg HBsAg using CFA as the adjuvant and received booster immunizations on the week 2 and week 6, using FA as the adjuvant. The eggs collected before immunization for 3 days were stored at 4℃and as control. The egg yolk was diluted 10 times by distilled water of pH=5, standed for 6 hours and then centriufged. The supernatant was precipitated with 55% and 33% ammonium sulfate respectively to obtain the crude sample. Then the curde sample was fractioned by gel filtration chromatogram on Sephadex G150, using the bueffer liquid (PH=7) which contained 0.02mol/L NaH2PO4-Na2HPO4 and 0.14 mol/L NaCl as eluant. The velocity of flow is 20 drops/min, and the volume of sample is 1mL. The first Peak is expected, and detemrined by SDS-APGE. Conclusion:the IgY is highly pure and the yield is 0.72 mgper milliliter yolk. The average molecule weight of IgY is 165KD. The titer of anibodyagainst HBsAg was 1/12800, determined by an enzyme linked immunosorbent assay (ELISA).New Double antibody Sandwich ELISA for HBsAg was developed, using anti-HBsAg mAb as capture antibody and HRP-labeled anti-HBsAg IgY as detection antibody. Anti- HBsAg IgY was coupled with HRP by facility prosodium iodate method. The result indicated that the rate of labeling was 54.31%, the titer of labeled antibody is 1:400. The optimal reaction conditions for the assay were determined. The result indicated that the optimal concentration of capture antibody was 1/4000; the working concentration of HRP-labeled IgY was 1:50; the coating conditions was 4℃overnight; the blocking solution was 1% defatted-milk-PBST; the HRP-IgY diluent was 1% defatted milk-PBST-4% PEG6000; the best time for the substrate was under room temperature for 15 minutes; the threshold for serum sample was 0.2; the lower detection limit of this ELISA was 2.5ng/mL. The intra- and inter-assay CV of DAS-ELISA is 4.72%,13.14%, respectivity. The cross-reaction indicated that the method is specific. Then, we detected 43 human serum by this new DAS-ELISA, and the results were consistent with commercial reagent kits.The results showed that we had successfully purified chicken anti-HBsAg IgY, gained HRP labeled chicken anti-HBsAg IgY and primarily established a new DAS-ELISA. We believed this DAS-ELISA assay would be a useful tool in the future HBsAg diagnosis.
|
PDF Full Text Request