Establishment and validation of a double antibody sandwich ELISA for the quantification of Coxsackievirus A10 antigen in multivalent hand, foot and mouth disease vaccines
10.13200/j.cnki.cjb.004712
- VernacularTitle:多价手足口病疫苗中柯萨奇病毒A组10型抗原含量双抗体夹心ELISA检测方法的建立及验证
- Author:
Junying GAO
- Publication Type:Journal Article
- Keywords:
Coxsackievirus A10(CV-A10);
Multivalent hand,foot and mouth disease(HFMD) vaccines;
Antigen content;
Double antibody sandwich ELISA
- From:
Chinese Journal of Biologicals
2026;39(06):694-703
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish and validate a double antibody sandwich ELISA method suitable for the detection of Coxsackievirus A10(CV-A10) antigen content in multivalent hand,foot and mouth disease(HFMD) vaccines in China,in order to apply it to the detection of CV-A10 antigen in multivalent HFMD vaccines.Methods According to the general chapter 9307 of the Chinese Pharmacopoeia(Volume Ⅳ,2025 edition),a conformational monoclonal antibody mAb5 with broad-spectrum binding and neutralizing activity targeting the receptor-binding domain was used as the detection antibody.A method for the detection of CV-A10 antigen content was established through analytical target profile(ATP) setting,risk identification,and Design of Experiments(DoE).The specificity,linearity,relative accuracy,and intermediate precision of the method were validated.The applicability of the method for CV-A10 vaccine bulks from six manufacturers was evaluated in combination with national antigen standards.Results Using the signal-to-noise ratio(S/N) as the response indicator,the optimal coating antibody concentration was determined to be 0.25 μg/mL,the detection antibody concentration 2 μg/mL,and the dilution factor of the enzyme-labeled antibody 1∶5 000.The method operable design region(MODR) for the incubation time of the antigen,detection antibody,and enzyme-labeled antibody was obtained using DoE,all ranging from 60 to 90 min,with a chromogenic time of 10 to 15 min.The optimal calculation model was the double logarithmic model.After validation,the established method demonstrated good specificity,a linear R~2 > 0.98,a relative accuracy with average relative bias ranging from-7.21% to 6.55%,and an intermediate precision with geometric coefficient of variation(GCV) ≤ 7.07%,all meeting the predefined ATP acceptance criteria.The applicability study results demonstrated good linearity and parallelism between the national antigen standard and the vaccine bulks from different manufacturers,suggesting that the established method was well applicable to CV-A10 vaccines from various virus strains and manufacturing processes.Conclusion A double antibody sandwich ELISA method for the detection of CV-A10 antigen content in HFMD vaccines was successfully established,which exhibits good specificity,rela-tive accuracy,and intermediate precision,making it suitable for the quantitative detection of CV-A10 antigen content in multivalent HFMD vaccines currently under development,thus providing an effective tool for vaccine quality control.
