Abstract
Objective To develop a double antibody sandwich ELISA method for quantitative determination of D antigen in poliovirus types Ⅰ, Ⅱ, Ⅲ and in vitro evaluation of Sabin strain inactivated poliovirus vaccine (sIPV) potency. Methods Bovine antisera and mouse monoclonal antibody (McAb) hybridoma cell lines were prepared from Simmental cattle and BALB/c mice immunized with purified D antigens, respectively. McAb ascites were prepared from BALB/c mice immunized with hybridoma cells. Purified bovine polyclonal antibodies (PcAbs) and mouse McAbs were obtained from bovine antisera and mouse ascites through precipitation and affinity chromatography, respectively. Double antibody sandwich ELISA method was developed using PcAb as coating antibody and McAb as detecting antibody after pairing screening. The method was verified for linearity, measurement range, specificity, accuracy, precision and applicability. Results Both PcAbs and McAbs had neutralizing activity, and their purities were more than 80% and 90%, respectively. McAbs 3H10, 901 and 1H10 were determined as detecting antibodies in quantitative ELISA for detection of typeⅠ, Ⅱ, Ⅲ D antigens, respectively, afer antibody pairing screen. The optimal concentrations of coating antibodies against type Ⅰ, Ⅱ, Ⅲ D antigens were 1∶8 000, 1∶8 000 and 1∶10 000, and of detecting antibodies 3H10, 901 and 1H10 were 1∶10 000, 1∶20 000 and 1∶10 000. The linear ranges of the method for type Ⅰ, Ⅱ, Ⅲ D antigen detection were 0.4-13.0, 0.4-12.0 and 0.4-20.0 DU/ml, with all coefficients of determination≥0.99.D antigens were specifically detected by the method and no cross reactions were found with C antigen, different type D antigen, and substances in production process. The ratios (×100%) of measured value to theoretical value of high, medium and low concentration samples were 97%-111%, 91%-104% and 95%-102%, with relative standard deviations ≤7%, ≤9% and ≤10%, respectively. The results reflected the trend of D antigen purification when using this method to measure samples from sIPV manufacture process. Conclusion The double antibody sandwich ELISA method for quantitative determination of D antigen in poliovirus typesⅠ, Ⅱ, Ⅲ is developed, and can be used for in vitro evaluation of sIPV potency. Key words: Poliovirus vaccine, inactivated; Enzyme-linked immunosorbent assay; D antigen; C antigen
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