Establishment and verification of an ELISA method for detection of porcine trypsin residues in reassortant rotavirus vaccine,live, oral, hexavalent (Vero cell)
10.13200/j.cnki.cjb.004653
- VernacularTitle:口服六价重配轮状病毒减毒活疫苗（Vero细胞）猪胰蛋白酶残留量ELISA检测方法的建立及验证
- Author:
Wenqi ZOU
1
Author Information
1. Department of Quality Control, Wuhan Institute of Biological Products Co., Ltd., Wuhan 430207, Hubei Province, China
- Publication Type:Journal Article
- Keywords:
Rotavirus(RV) vaccine;
Porcine trypsin;
ELISA;
Quality control
- From:
Chinese Journal of Biologicals
2026;39(02):189-194+200
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish an ELISA method for the determination of porcine trypsin residues in reassortant rotavirus vaccine, live, oral, hexavalent(Vero cell) and verify the method, so as to control the quality of the products more comprehensively.Methods The absorbance values of standard and test solutions were determined at 450 nm. The concentration value of standard was used as independent variable X and the mean value of the absorbance value was used as dependent variable Y to establish a four-parameter linear regression equation. The porcine trypsin content of the test solution was calculated using the linear regression equation. The method was verified for linear range and detection limit, accuracy, precision,robustness and specificity. Results Porcine trypsin concentration showed good linearity in the range of 1. 56-100 ng/mL(R~2> 0. 99). The coefficients of variation(CVs) of single virus harvest solution and monovalent bulk solution in repeatability verification was 5% and 8%, and the CVs of single virus harvest solution and monovalent bulk solution in intermediate precision verification was 5% and 6%, respectively. In detection limit verification, the recovery rate was 102%-112% at the porcine trypsin concentration of 1. 56 ng/mL, with the CV of 4% and the quantification limit of 1. 56 ng/mL. In specificity verification, the recovery rates of the test solution diluted with bovine serum albumin(BSA) were 101%-102%, and the recovery rates of the test solution diluted with sucrose-phosphate-glutamate(SPG) buffer and DMEM were 90%-101%,indicating the excipients had no interference with the detection. The CVs of the results at different color development time and temperatures for robustness verification were less than 15%. In accuracy verification, the recovery rates of single virus harvest solution and monovalent bulk solution were within 86%-105% and 109%-117%, respectively.Conclusion The linear range and detection limit, accuracy, precision, robustness and specificity of the established ELISA method were all in line with the acceptance criteria and were suitable for the determination and quality control of the residual amount of porcine trypsin in reassortant rotavirus vaccine, live, oral, hexavalent(Vero cell).
