Establishment Of Heptitis B Virus Core Antigen Double Antibody Sandwich Method

BackgroundHeptitis B virus infection is a major public problem in the world.WHO estimates that globally,257 million people have been chronically infected with HBV.Recent finding demonstrate that HBV infection is hardly to be completely cured,but a functional cure can be achieved.On the one hand,after antiviral therapy,virus replication was inhibited in patients with HBV infection.In some patients who had been treated with NAs and then treated with IFN-α,the antiviral effect was better,HBV DNA was under the detection limit,and the level of HBsAg decreased or even could not be detected,reaching the functional cure standard.On the other hand,children with chronic hepatitis B not only tend to be HBsAg negative,but also produce protective antibodies after interferon therapy.Therefore,how to evaluate the functional cure of HBV is particularly important.HBV DNA and other HBV serological indicators cannot evaluate HBV functional cure.It is worth noting that HBV DNA in patients with antiviral therapy is often undetectable,integration of the HBV genome will still produce HBsAg and viral mutations often lead to HBeAg negative.In recent years,HBV pgRNA and HBcrAg have been proposed to monitor the number and transcriptional activity of intrahepatic ccc DNA,which can predict the risk of recurrence after drug withdrawal.HBcrAg contains HBcAg,HBeAg,and P22.The serum concentration of HBeAg is much higher than HBcAg.Therefore HBcrAg cannot play an evaluation role in HBeAg（+）and HBeAg（-）patients.HBcAg is a viral nucleocapsid protein that is translated from pgRNA and is not directly affected by antiviral drugs.The quantitative detection of HBcAg in serum can play an evaluation role in HBeAg（+）and HBeAg（-）patients,predict the response of antiviral therapy patients and the risk of relapse after drug withdrawal.It has the potential to be an evaluation index for HBV functional cure,and it is meaningful to study HBcAg detection.In order to obtain high affinity antibodies,we chose to prepare rabbit mAbs.First,HBcAg was synthesized as an immunogen,followed by immunization of New Zealand rabbits,and finally 4 mAbs were obtained.In order to obtain antibodies that specifically recognize HBcAg,we synthesized HBcAg-C-terminal specific peptides as immunogens,immunized BALB/c mice,and obtained 5 mAbs later.The obtained antibodies were identified by ELISA and Western blot.Using biotin labeling technology to further improve the monoclonal antibody recognition ability.We selected mAbs with strong specificity for pairing to initially establish the HBcAg double antibody sandwich ELISA method.Objective1.Screening rabbit mAbs that recognize HBcAg;2.Preparation of mouse against mAbs HBcAg-C terminal specific sequence;3.Preliminary development of double antibody sandwich ELISA method to detect HBcAg.MethodsRecombinant HBV core antigen was expressed in Ecoil BL21（DE3）,and HBcAg particles in soluble fraction were purified by Affinity chromatography.Four New Zealand rabbit were immunized with rHBcAg and then rabbit serum were collected and detected by indirect ELISA.The immunized rabbits with high immune titer were selected,and the spleen cells were fused with 240E-W2 myeloma cells.After 15 days of culture,the supernatant of cell culture was collected.After 15 days of culture,the supernatant of cell culture was collected.The stable positive monoclonal cell lines were identified by ELISA and western blot.The C-terminal specific polypeptide sequence of HBcAg was designed and synthesized,and the polypeptide was used as the immunogen to immunize BALB/c mice.One week after 3^rd immunization,blood was collected from the tail vein of mice,and the titer of mouse antiserum was detected by indirect ELISA method.The immunized mice with high immune titer were selected,and the spleen cells were fused with SP2/0 myeloma cells.After 10 days of culture,the supernatant of the cells was collected.The stable positive monoclonal cell lines were identified by ELISA.Ascites was obtained by in vivo induction in mice and the antibody was purified.Results1.Expression of rHBcAgThe rHBcAg were successfully expressed in Ecoli BL21（DE3）and verified by sequence analysis.The sizes of rHBcAg was about 21 KDa by SDS-PAGE and western blot.2.Rabbit antiserum titerThe pre-immune serum and antiserum after the 3^rd immunization in an limiting dilution by ELISA.Four rabbit antiserum all could reach 512000,which the#4 rabbit was the highest.3.Results of the fused hybridoma cellsSplenocytes from the two immunized rabbit were fused with myeloma cells.The fused hybridoma cells were selected in direct ELISA.In the first round of screening of 450 96-well plates,90 cell strains supernatant was detected that the value of OD_450nm was beyond 0.5.After second round of screening of 90 cell strains,we obtained 7 stable positive cell strains.4.Specificity identification of rabbit monoclonal antibodies against HBcAgAt 0.4μg/ml recombinant HBcAg coating well by ELISA,the 7 anti-HBcAg mAbs obtained above were tested and diluted to 4000 times.The results of OD_450nm50nm values were all>0.5,indicating that the 7 antibodies can effectively recognize rHBcAg.The#1,#3 mAbs diluted to 16000 times,the results of OD_450nm50nm value was still>0.5,and the negative control<0.1,indicating that the two strains have stronger binding activity to rHBcAg.As a result of western blot,when antigen was HBV infectious cells,the strain 5,6,7,and 8 were positive.When antigen was rHBcAg,the strain 1,3,5 and 7 were positive.5.Antibody sequence alignment analysisSeven of the mAbs were sequenced,but 4 mAbs were the same sequence.6.Identification of anti-HBcAg-C terminal mouse m Ab by indirect ELISAFive mAbs were prepared by immunizing mice with HBcAg-C-terminal specific peptides.At 0.3μg/ml HBcAg-C-terminal peptide-1 coating well with ELISA,the No.2 mouse mAb was diluted 16000 times and the results of OD_450nm50nm value was 0.179,indicating that the No.2 mouse mAb can effectively recognize HBcAg-C-terminal polypeptide-1.At 0.3μg/ml HBc-C-terminal peptide-1 coating well with ELISA,the No.5 mouse mAb was diluted to the same multiple,and the result of OD_450nm value of 0.292,indicating that the No.5 mouse mAb can effectively recognize HBcAg-C terminal polypeptide-2.At 10μg/ml r HBcAg coating well with ELISA,0.625μg/ml No.2 mouse mAb as the primary antibody,and the results of OD_450nm value was 0.236,indicating that the No.2 mouse mAb can effectively recognize rHBcAg.7.Indirect ELISA detection of biotin-labeled mouse mAb against HBcAg-C terminalIn order to improve the detection sensitivity,the No.2 mouse mAb was labeled with biotin.At 10μg/ml rHBcAg coating well with ELISA,50μg/ml the biotin-labeled No.2 mouse mAb as the primary antibody,the results of OD_450nm value was close to2.0,indicating that the biotin-labeled No.2 mouse mAb can effectively recognize rHBcAg.8.Preliminary construction of HBcAg method for double antibody sandwich ELISA detection.We tried different rabbit polyclonal antibodies/rabbit mAbs as coating antigens,the biotin-labeled No.2 mouse mAb as secondary antibody,and established a double antibody sandwich ELISA method to detect rHBcAg.Rabbit polyclonal antibody was used as the coating antibody,the concentration was diluted from 1:100 to 1:2500,different concentrations of rHBcAg were detected,and 2μg/ml the biotin labeled No.2 mouse mAb as secondary antibody,3.2 ng/ml rHBcAg was detected,the results of OD_450nm value was 0.2～0.5,and negative control value were<0.15,indicating that this combination can effectively recognize rHBcAg.Furthermore,the biotin-labeled No.2 mouse mAb can effectively recognize rHBcAg.Conclusions1.The rHBcAg was successfully expressed in E.coil BL21（DE3）and purified.2.Immunization of New Zealand rabbits,rabbit anti-HBc Ag serum titers all have reached a high level,indicating that the immunization strategy is successful.Using the hybridoma technology,4 rabbit mAbs were obtained and,which can effectively recognize rHBcAg.3.Immunized mice,obtained 5 mouse mAb which aimed at specific C-terminal of HBcAg by hybridoma technology.The No.2 mouse mAb can effectively recognize HBcAg-C-terminal peptide-1 and rHBcAg.The No.5 mouse mAb can effectively recognize HBcAg-C-terminal peptide-2.ObjectiveTo recombinant and purified rabbit mAbs for the development of HBcAg double antibody sandwich ELISA.MethodsThe rabbit mAbs target gene fragment was recovered and cloned into the pcDNA3.0 vector to construct the expression plasmid.The recombinant expression plasmid was transfected into 293T cells,and the expression supernatant was obtained and purifiedby protein A.The specificity was identified by ELISA method.2 rabbit mAbs were labeled with biotin to improve the sensitivity of detection.The rabbit mAbs and mouse mAbs were combined to construct a double antibody sandwich ELISA method to detect HBcAg.Results1.The expression plasmids of 4 mAbs were successfully constructed by agarose gel electrophoresis and sequencing analysis.The heavy chain expression plasmid and light chain expression plasmid were co-transfected into 293T cells.After transfection,the supernatant was purified by protein A and analyzed by Coomassie brilliant blue staining,and the results showed that all the 4 recombinant antibodies could be expressed successfully.2.Rabbit mAb#1 and#5（2μg/ml）were mixed as coating antibodies,10μg/ml the biotin-labeled NO.2 mouse mAb as the recognition antibody.Regardless of any condition,the results of OD_450nm values all were<0.4,negative control value was0.20～0.25.Under the same coating condition,0.5μg/ml biotin-labeled rabbit mAb#3 and#7 were mixed as secondary antibody.The detection of 40 ng/ml rHBcAg OD_450nm value was 0.13,and the negative control OD_450nm value<0.05,indicating that this combination can be detected the lowest concentration of rHBcAg was 40ng/ml.Conclusion1.Successfully constructed a eukaryotic expression system that can express a large amount of rabbit monoclonal antibody.The mAbs all had strong binding activity with rHBcAg.2.Initially established a sandwich ELISA method for detecting HBcAg double antibody,which is based on rabbit mAb and detected rHBcAg to reach ng quantity.