Development and verification of a double-antibody sandwich ELISA method for quantitative determination of hepatitis A virus based on specific monoclonal antibody preparation
10.13200/j.cnki.cjb.004761
- VernacularTitle:甲型肝炎病毒单克隆抗体的制备和抗原定量双抗体夹心ELISA检测方法的建立及验证
- Author:
Tingting FU
- Publication Type:Journal Article
- Keywords:
Hepatitis A virus(HAV);
Monoclonal antibody(mAb);
Double-antibody sandwich ELISA
- From:
Chinese Journal of Biologicals
2026;39(09):1079-1085
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare monoclonal antibodies(mAbs) against the hepatitis A virus(HAV) L-A-1 strain using mouse hybridoma technology,establish and verify a double-antibody sandwich ELISA for HAV antigen quantification,so as to provide an effective detection method for monitoring antigen content during vaccine production.Methods Using HAV L-A-1 as the immunogen,five female BALB/c mice were immunized to generate monoclonal antibody hybridoma cell lines through mouse hybridoma cell fusion technology.Ascites was prepared by in vivo induction in mice,and monoclonal antibodies were purified by Protein A affinity chromatography.The monoclonal antibodies were determined for the titers by indirect ELISA,analyzed for the purity by 12% SDS-PAGE,verified for the specificity by Western blot,and measured for the relative affinities by indirect ELISA.Two monoclonal antibodies with higher relative affinities were selected as coating antibody and enzyme-labeled antibody,respectively,and the optimal working concentrations were determined by checkerboard titration to establish a detection protocol.The method was validated for specificity,lower detection limit,linear correlation,accuracy and precision,and was preliminarily applied to the analysis of three batches of test samples.Results Four HAV hybridoma cell lines were successfully screened: 2G3,5F6,4E2,and 2A4.After purification,the titers were measured to be 10~(-6)-10~(-7),with all the purity exceeding 95%.All the four monoclonal antibodies could specifically bind to HAV VP1 protein,and the relative affinities were ranked as follows: 2A4 > 5F6 > 4E2 > 2G3.Using 2A4 as the coating antibody and5F6 as the enzyme-labeled antibody,the optimum working concentrations were 10 μg/mL and 2 μg/mL,respectively.The method detected no cross-reactivity with human diploid cell supernatant,newborn bovine serum and human serum albumin.The lowest detection limit for HAV antigen was 0.136 7 IU/mL,with a linear range of 0.136 7 to 8.75 IU/mL,R~2> 0.97.The accuracy validation showed a recovery rate of 92.7% to 103.0% for viral antigen detection.The intra-assay and interassay coefficients of variation(CVs) in precision verification were 0.89%-2.27% and 0.33%-2.65%,respectively.The antigen content in three batches of test samples showed a linear correlation with the dilution factor.Conclusion The HAV mouse monoclonal antibodies were successfully prepared and the double-antibody sandwich ELISA method was established,which was stable and could accurately detect the antigen content,providing effective data for the control of antigen content in the production of hepatitis A vaccine.
