| Hepatitis B is an infectious disease,which is caused by Hepatitis B virus.It is one of the most serious and common health problems in the world.HBV infection can cause a variety of liver damages including acute self-healing infections,chronic hepatitis,fulminant hepatitis,cirrhosis and liver cancer.Currently,HBV is divided into four major serotypes as "adw","adr","ayw" and "ayr" based on HBsAg(Hepatitis B Surface Antigen)protein molecules and nine genotypes including A-I genotypes by the genomic nucleotide sequence differences.Many studies showed that HBV serotypes and genotypes led a severe effect on the progression of hepatitis B disease,such as clinical manifestations,the interferon treatment response and its prognosis.Many studies found that both HBV serotype and genotype obviously had the geographic distribution characteristics.The research on such characteristics could give much help for HBV clinical outcome,HBV antiviral efficacy and SNP(single-nucleotide polymorphism)of HBV gene mutations,and also have an important role in the study of the biological properties of HBV.This study focused mainly on the following three aspects:1.Establishment of the cell lines for HBsAg antigenic determinantsThe Balb/C mice were immunized with different HBsAg subtype proteins.The titers ofmice antisera(pAb or polyclonal antibodies)after the special immunizations with HBsAg subtype proteins ad,adr and adw reached much higher level.After the fusion experiments and HTS-ELISA,four hybridoma cell lines which could stably secret monoclonal antibodies were screened out,and named as#2.1-4.2,#18.1-23-3.1,#7-7-2,and#56.2-71-5.2,respectively.2.Identification of the monoclonal antibodies anti-HBsAg epitopesThe antibodies were purified by Protein-A affinity chromatography after the four hybridoma cell lines were cultured on MD6 medium.To analyze the purity,affinity,specificity and epitope characteristics of the monoclonal antibodies,it was found out that,#2.1-4.2,#7-7-2,#18.1-23-3.1 and#56.2-71-5.2 could recognize HBsAg "d,y,r,w" epitopes,respectively,through the comparison the specificity of these four monoclonal antibodies.Furthermore,direct competition ELISA demonstrated that#2.1-4.2 vs#7-7-2 and#18.1-23-3.1 vs#56.2-71-5.2 didn’t compete each other to the epitopes of HBsAg.All of the four antibodies had high purity(over 98%)and high affinity(over 109 L/mole).The heavy chain subtypes of#2.1-4.2,#18.1-23-3.1 and#56.2-71-5.2 were IgG2b,and#7-7-2 was IgGi.The light chain subtypes of all of these mAbs were κ.To identify the specific epitopes,four peptides were designed and synthesized based on amino acid sequences of HBsAg epitopes.The results of indirect ELISA showed that the antigen site of#18.1-23-3.1 might be similar to R1 peptide sequence,and the antigen site of#56.2-71-5.2 might be similar to W1 peptide sequence.3.Establishment of the sandwich ELISA method for HBV serotypingTo detect HBsAg serotype by sandwich ELISA,four monoclonal antibodies were labeled with HRP,separately and the average optimal dilution ratio(LD50)of these HRP labeled mAbs were 1:8,000.The sandwich ELISA was established through using#3-11-2.1(Anti-HBsAg"a"epitope)as the coating antibody while the HRP labeled mAb as the secondary antibody.The optimal coating concentration in this sandwich ELISA for#3-11-2.1 was 1.0 μg/mL;the optimal volume of sera samples was 10 μL;the optimal reaction temperature was 37℃;the reaction time was 30 minutes.One-step ELISA detection method had more accurate than two-step detection method.The evaluation of HBV serotype detection method was as follows:(1)The method had high specificity to identify four epitopes by specific capture HBsAg ELISA.(2)The detection method was established and the data showed a good liner range with R values.The coefficient variation of "intra-assay" was controlled less 10%and the coefficient variation of "inter-assay"was about 15%or below.(3)This method had no significant differences compared with PCR method.4.Analysis of the relationship among the distribution of HBV serotype,the HBVgenotype and the clinical outcome of HBVHBV sera samples of hepatitis patients were collected within China and the serotypes and genotypes of these samples were successfully analyzed.The data of 408 cases of HBV positive samples showed 330 cases were the adr serotype;50 cases were the adw serotype;and 14 cases had both ayw and ayr serotypes,respectively.In HBsAg genotyping,there were 116 cases of B genotype;255 cases of C genotype;37 cases of D genotype;and A,E,F,G,H genotypes were not found.For the differences of serotypes and genotypes,the distribution of gender in serotypes and genotypes had no significant difference,and the distribution of age had no significant difference as well.At HBV DNA level,ALT level and HBeAg positive rate had a significantly difference in the samples between serotype and genotype.The serotype adr had the highest level of HBV DNA and ALT,and then followed by adw and ayr,the ayw had the lowest level.HBsAg serotype adw had the highest positive rate of HBeAg,and then followed by adr,ayw and ayr in series.HBV genotype C had the highest HBV DNA level,ALT levels and HBeAg positive rate in the serum.Both HBV DNA and ALT level for the HBV genotype D were higher than genotype B,while the HBeAg positive rate in these genotypes was not.The distribution of HBV serotypes and genotypes from these patients and heathy patients was significantly different in our country.The adr serotype was the major HBsAg serotype nationwide in China and the adw was mainly located in the south of China.The serotype ayr and ayw were mainly distributed in Xinjiang region.The genotype C was the genotype predominantly in various regions of our country.The genotype B showed a concentrated distribution in the areas of eastern,central and southern of China and the genotype D existed mainly in Xinjiang area.In this study,both HBV serotype and genotype for HBV patient sera samples were performed simultaneously by the exclusive methods partially established,which could make the results more representative and reliable.The data could reflect the characteristics of HBV serotype and genotype distribution with much accuracy.These research findings in this study will provide a powerful tool for the research of HBV infection,a great guidence for clinicians to recognise HBV pathogenesis,epidemiology and the prognosis,diagnosis,treatment and prevention of Hepatitis B disease. |
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