Establishment and application of sandwich ELISA for detection of specific antigen of Seoul virus L99 strain / 中华微生物学和免疫学杂志

ABSTRACT

Objective:To establish a double antibody sandwich ELISA for detecting the specific antigen of Seoul virus (SEOV) L99 strain and to provide a means for antigen detection in the development, production and verification of vaccine against hemorrhagic fever with renal syndrome (HFRS).Methods:Monoclonal antibodies (McAbs) aganist L99 virus were induced in mice using four hybridoma cell lines and purified by Protein-A affinity chromatography. The purity, titer and specificity of McAbs were determined by SDS-PAGE, indirect ELISA and Western blot, respectively. Four McAbs were paired with each other and the additivity indices of paired McAbs were analyzed. After labeling McAbs with horseradish peroxidase (HRP), the concentrations of the coated and labeled antibodies were optimized by orthogonal test, and then a double antibody sandwich ELISA for virus antigen detection was established. Type Ⅱ HFRS inactivated vaccine standard was used as a quantitative standard to verify the sensitivity, linearity, specificity, accuracy and precision of the developed method. The applicability of the method was verified by testing three batches of vaccine stock solutions.Results:Four McAbs were at titers of greater than 1∶10 6 and their purity was all greater than 98%. The McAbs secreted by 1D5, 3A4 and 5B7 cells could specifically recognize the nucleocapsid protein of SEOV L99. There was cross-reaction between McAb secreted by 1D5 cells and Hantaan virus PS-6. The McAbs secreted by 3A4 and 1D5 were used as coating and labeling antibodies based on the results of antibody pairs. The working concentrations of the coating antibody and the horseradish peroxidase (HRP)-labeled antibody were 20 μg/ml and 1∶4 000, respectively. The minimum detection limit of the established method for the detection of SEOV L99 antigen was 0.078 1 μg/ml, and the linear range was 0.078 1-2.500 0 μg/ml with a R2 value of more than 0.99. There was no cross reaction with other HFRS vaccine. The virus antigen recovery rate was between 95.8% and 108.7%, and the coefficients of variation of precision was less than 10%. Three batches of Type II HFRS inactivated vaccine stocks were detected by this method and the results was dose-dependent. Conclusions:This study successfully established a double antibody sandwich ELISA method for specific detection of SEOV L99 strain antigen in the production of bivalent HFRS vaccines produced from hamster kidney cells.