高效体积排阻色谱检测口蹄疫灭活疫苗中146S抗原含量的疫苗前处理方法比较

Comparison of Different Demulsification Methods for Quantification of 146S Antigen in Inactivated Foot-and-Mouth Disease Virus Vaccine by High Performance Size Exclusion Chromatography (HPSEC)

关键词:  口蹄疫  灭活疫苗  146S  高效体积排阻色谱  破乳

Key words:  foot-and-mouth disease(FMD)  inactivated vaccine  146S  high performance size exclusion chromatography(HPSEC)  pretreatment

摘要:

为了提高高效体积排阻色谱(HPSEC)检测口蹄疫灭活疫苗146S抗原含量的准确性、适用性和操作性,分别采用正戊醇、正丁醇和三氯甲烷对9批口蹄疫灭活疫苗进行前处理,对三种方法的破乳后水相得率和146S抗原含量的色谱检测结果进行比较。结果表明,破乳后水相得率分别为：正戊醇38%~49%,正丁醇42%~44%,三氯甲烷50.0%；9批疫苗的色谱结果均检测到146S特征峰,146S含量在0.7μg/mL~12.5μg/mL,三次测定的相对标准偏差最大值为4.0%,9批疫苗不同前处理方法的检测结果均值具有统计学意义,三氯甲烷组与正戊醇组数据一致性强,正丁醇组测定值偏低。本实验进一步优化了口蹄疫灭活疫苗146S抗原含量检测的高效体积排阻色谱法,为该方法的科学应用提供了数据支撑。

Abstract:

This study is aimed at improving the accuracy, applicability and operability of quantification of 146S antigens in foot-and-mouth disease (FMD) inactivated vaccine by high performance size exclusion chromatography (HPSEC). Nine batches of FMD inactivated vaccines were pretreated with n-amyl alcohol, n-butanol and trichloromethane respectively. Extraction rate of water phase after demulsification and chromatographic detection results of 146S antigens were compared. The results of the extraction rates of aqueous phase were shown as following: n-amyl alcohol 38%~49%, n-butanol 42%~44%, trichloromethane 50.0%. The peak of 146S antigen was detected in chromatographic results within all 9 batches of vaccines, with the content ranging from 0.7μg/mL to 12.5μg/mL. The maximum relative standard deviation of the three measurements of each pretreatment was 4.0%. The mean value of the detection results of the 9 batches of vaccines with different pretreatment methods was statistically significant. The trichloromethane group showed a strong consistency with the n-amyl alcohol group, while the n-butanol group had a low level of the 146S antigen content. This study further optimized the HPSEC on quantification of 146S antigens in FMD inactivated vaccines, which also provided data support for the scientific application of the method.