Development, verification and preliminary application of a quantitative double antibody sandwich ELISA for Coxsackievirus A6 antigen
10.13200/j.cnki.cjb.004338
- VernacularTitle:柯萨奇病毒A组6型抗原含量双抗体夹心ELISA检测方法的建立、验证及初步应用
- Author:
LU Weiwei
- Publication Type:Journal Article
- Keywords:
Coxsackievirus A6(CV-A6);
Antigen content;
Enzyme linked immunosorbent assay(ELISA);
Tetravalent handfoot-mouth disease vaccine
- From:
Chinese Journal of Biologicals
2024;37(11):1341-1348
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a double antibody sandwich ELISA for the determination of the Coxsackievirus A6(CVA6) antigen content, and to verify and preliminarily apply it for the CV-A6 production process and the quality control of tetravalent hand-foot-mouth disease(HFMD) vaccine.Methods Paired antibodies were screened and double antibody sandwich ELISA was developed by using purified CV-A6 sheep polyclonal antibody as coating antibody and mouse anti-CVA6 monoclonal antibody as detection antibody. The experimental conditions were optimized, and the linearity, range,specificity, accuracy, precision and robustness of the method were verified. The developed method was used to detect the antigen content of CV-A6 antigen production process samples and final products.Results Both of the sheep polyclonal antibody and monoclonal antibody 16D1-1E5 had neutralizing antibody activity, while the other three monoclonal antibodies showed no neutralizing antibody activity. The ELISA titers of all antibodies were not lower than 1. 00 × 105. The monoclonal antibody 2B6-4F9 was IgG3 subtype, while the other three monoclonal antibodies were IgG2a subtype. Using sheep polyclonal antibody as coating antibody and monoclonal antibody 16D1-1E5 as detection antibody for pairing, the method was developed.The dilution ratios of coating antibody and detection antibody were 1∶2 000-1∶6 000 and 1∶4 000-1∶6 000 respectively.The linear range of the developed method was 0. 664-21. 263 U/mL, with the R2≥ 0. 99. The method could only detect CVA6 antigen specifically and had no cross-reaction with CV-A10, CV-A16 and enterovirus 71(EV-71) antigens. M199,DMEM, Vero cell protein and bovine serum protein probably existing in the production process had no interference with this method. The measured/theoretical values of samples with high(21. 00 U/mL), medium(10. 50 U/mL) and low(1. 70 U/mL)concentrations were between 95% and 105%, with the RSDs within 15%. In the range of 0. 664-21. 263 U/mL, the R2was not less than 0. 99, the measured/theoretical values were between 95% and 105%, and the RSDs of precision verification was within 15%. The method had no difference in accuracy and precision at different incubation temperatures(35, 37, 39 ℃).The antigen content of CV-A6 antigen production process samples and finished products detected by the developed method effectively reflected the changing trend of antigen concentration in process samples and final products.Conclusion The double antibody sandwich ELISA for quantitative determination of CV-A6 antigen was developed with good linearity, range,specificity, accuracy, precision, and robustness, which can be used for the quality control of CV-A6 antigen process and final products, and lays a foundation for the in vitro efficacy evaluation of CV-A6 antigen.
