Sabin株脊髓灰质炎灭活疫苗D抗原含量检测方法的建立

Development of a detection method for the D antigen content of Sabin strains inactivated poliovirus vaccines

目的 建立检测Sabin株脊髓灰质炎灭活疫苗中D抗原含量的双抗体夹心ELISA方法。方法 采用纯化的D抗原制备鼠单抗和牛多抗,采用微量中和病变和间接ELISA检测抗体的效价与特异性。以牛多抗为包被抗体、小鼠单抗为显示抗体建立夹心ELISA检测D抗原含量的方法,并进行方法学验证。结果 制备了高效价的Ⅰ、Ⅱ、Ⅲ型牛多抗和鼠单抗,牛多抗微量中和效价分别为409 600、96 000和128 000,鼠单抗微量中和效价分别为256 000,128 000和256 000,并可与相应型别D抗原特异性结合。采用这些抗体成功建立了夹心ELISA方法,3个型别标准曲线线性良好,R^(2)>0.99。对所建立的方法进行验证,其结果显示,Ⅰ、Ⅱ、Ⅲ型加标回收率分别为80.4%~117.7%、90.3%~111.6%、91.9%~111.3%,均在80%~120%范围内;实验内与实验间CV均<10%;并且能够有效区分D抗原与C抗原。采用国内5家企业生产的疫苗进行了适用性验证,结果显示方法适用性良好。结论 成功建立了检测Sabin株脊髓灰质炎灭活疫苗中D抗原含量的双抗体夹心ELISA方法,该方法具有良好的准确性与精密度,特异性强,并且能够检测不同企业生产的疫苗。

Objective To develop a sandwich ELISA method to detect the D antigen content of Sabin strains inactivated poliovirus vaccines(sIPV). Methods The bovine polyclonal and mouse monoclonal antibodies were obtained by immunizing the animals with each type of sIPV, respectively. The titers and specificities of each antibody were tested by indirect ELISA and microneutralization assay. A sandwich ELISA test to determine the D antigen content in sIPV was developed in which the purified bovine antibodies were used as capture antibody and mouse monoclonal antibodies as detection antibody. The validation of the method included accuracy, precision and specificity. Results The antibodies with higher titers to each type sIPV were successfully obtained. The neutralizing titers of bovine polyclonal antibodies were 409 600、96 000 and 128 000 for type Ⅰ,Ⅱ and Ⅲ respectively. The neutralizing titers of monoclonal antibodies were 256 000,128 000 and 256 000 for type Ⅰ,Ⅱ and Ⅲ respectively. These antibodies could specifically bind to their corresponding D antigens. A sandwich ELISA using these antibodies for determination of D antigen was thus established. The recovery rates for typeⅠ,Ⅱ and Ⅲ D antigens were 80.4%-117.7%, 90.3%-111.6% and 91.9%-111.3% respectively, which were all within the range of 80%-120%. The CVs of intra-and inter-assays were below 10%. This test could also distinguish D from C antigens. The D antigen contents of 10 lots of sIPV from 5 Chinese companies were measured by the established method and the results showed that the method was applicable to all these vaccines. Conclusion A sandwich ELISA method for determination of D antigen content in sIPV was successfully established with satisfying sensitivity, accuracy, precision and specificity, and moreover, it was applicable to products from different companies.