Monoclonal Antibody Preparation, Epitope Phenotyping And Clinical Application Of Hepatitis B Surface Antigen (HBsAg)

Serological HBV markers are used in clinical practice of the prognosis and disgnosis of HBV infection and may be used for the guidance of Hepatitis therapy. Hepatitis B surface antigen (HBsAg) is one of the most common HBV infectious markers in the clinical field. The marker could be detected in 2-8 weeks from the HBV infection to the onset of jaundice and the manifestation of liver dysfunction. HBsAg is the best candidate to detect HBV infection in blood check up and clinical diagnosis, which is coded by HBV S gene. According to the difference of antigen epitopes, HBsAg is mainly divided into four subtypes, e.g. adw, adr, ayw and ayr. The epitope "a" exists in all HBsAg subtypes, while two epitope pairs of the d/y and the w/r are mutually exclusive in the HBsAg subtypes. The subtype of HBsAg shows a geographic distributuion significantly, which is related to the disease development. Clinical significance of HBsAg subtype has become a hot topic in the research of HBV. In the detection of HBV and the identification of HBsAg subtype would provide a great value in the prognosis, diaganosis and treatment of Hepatitis. Among these HBsAg subtypes, the epitope "a" becomes the best target for Hepatitis B diagnosis and vaccine design.Based on the specific reaction of antigen and antibody, ELISA is currently the most important method for HBsAg detection and is broadly used in the clinical field. It is less cost, easily to handle and often be applied to the large scale screening (HTS). Recently, more and more new methods and technologies are appeared in the HBsAg assays, including microparticle enzyme immunoassay, chemiluminescence assay, and flurecence-gold nanoparticle immunoassay etc. In general, monoclonal antibody with the specificity and high sensitivity is the basic element for the technology development.In this study, I discussed the preparation and application of high sensitivity monoclonal antibody aganist HBsAg. Meanwhile, I made a priliminary research on the preparation of HBsAg subtype monoclonal antibody. The processes of my research are showed as below.1. The preparation of HBsAg common determinant "a" specific monoclonal antibody. Two groups of Seven weeks old Balb/C mice were immunized by HBsAg-ay and HBsAg-ad antigen respectively. The dose dependent titration ELISA was taken for those immunized mice and showed they all have certain level of HBsAg ay or ad immunal responses. I have chosen the spleen cell donors with higher titers, and then fused with myeloma cell SP2/0 in PEG solution. After a large scale screening and hybridoma subclones, I have selected 5 stable antibody-secreting hybridoma in which are HBsAg specific. The specificity of 3 hybridoma were showed to the common determinant "a" and named #2-11-2,#3-16-1 and #6-2-2 mAbs. 2. The assays of "a" specific mAbs and the mAb application. The characteristics of "a" mAbs are:(1)#3-11-2, IgG2a andκ(kappa) light-chain; (2)#6-2-2, IgGl andκ(kappa) light-chain. I have labeled #3-11-2 mAb with HRP for the competition ELISA of mAb binding to the epitope. I chose a pair of mAbs against the different epitopes with high specificity and affinity, and established an HBsAg detection ELISA. In this assay,0.8μg/mL and 100μL/well of #6-2-2 was coated on the 96-well ELISA plate and the detecton antibody was used by #3-11-2-HRP with 1:10,000 dilution. Considering the validity and sensitivity of this assay, the positive control samples from commercial kit were used as the samples, and the results showed that the samples were detected efficiently. In further, this HBsAg assay was applied in clinical samples in cluding normal and HBV patients'samples. The comparison of our method of this assay and the HBsAg detection method in the hospitals (e.g. Roche Modular E170), my system has better repetitiveness, linearity and great matches to the data from clinical assays.3. The priliminary research on the preparation of mAbs against HBsAg subtypes. I have explored these HBsAg mAbs to define the specificity of HBsAg subtype "y" and "d", which the mAbs were harvested from the determinants "ad" and "ay" screening ELISA. If the mAb has no specificity or low affinity to common determinant "a", but it has either an HBsAg-ad or an HBsAg-ay high affinity, I would consider the mAb was indicated HBsAg "y" or "d" epitope specific. In this study, I have suceecefully harvested two HBsAg "y" epitope specific monoclonal antibodies.