流感疫苗血凝素含量测定替代方法的建立及验证

目的建立测定流感疫苗血凝素(Haemagglutinin,HA)含量的替代方法 ,并进行验证,以解决大流行流感发生初期疫苗原液中HA定量的难题。方法优化糖苷酶处理条件,将流感疫苗原液经糖苷酶处理后,以还原SDS-PAGE方法分离样品,以灰度扫描法确定HA蛋白百分比,结合样品总蛋白数值,计算样品中HA含量。以该方法和传统的单向免疫扩散(SRID)法分别测定7批流感疫苗原液中HA含量,并进行比较。结果优化的糖苷酶处理条件为:总蛋白400μg/ml,PNGaseF加入比例为1:50。样品经糖苷酶处理后,以还原SDS-PAGE法可以清晰区分各蛋白条带,经测序后确定HA两个亚基,与预期相对分子质量一致。该方法测定7批流感疫苗原液中HA含量的结果与SRID法测定结果的符合率在87.90%~122.20%之间。结论初步建立了测定流感疫苗中HA含量的替代方法 ,在WHO参考品供应不到位时,可用于大流感发生时疫苗原液中HA之定量。

Development and Validation of Alternative Method for Determination of Haemagglutinin Content in Influenza Vaccine

Objective To establish an alternative method for determination of haemagglutinin(HA)content in influenza vaccine so as to solve the problem of quantitative determination of HA in vaccine bulk at early stage of outbreak of pandemic influenza.Methods Influenza vaccine bulk was treated with PNGase F under an optimized condition,and separated by reduced SDS-PAGE,in which the percentage of HA protein was determined by densitometric analysis.The HA content was calculated based on the percentage of HA and total protein content in test sample.The HA contents in seven batches of vaccine bulks were determined by the alternative method and traditional SRID method respectively,and the results were compared.Results The optimal total protein content of bulk for treatment with PNGase F was 400 μg /ml,and the optimal ratio of PNGase F to bulk was 1:50(v /v).After treatment,various protein bands in test samples were differentiated clearly by reduced SDS-PAGE,of which two HA subgroups were identified by sequencing,with the identical relative molecular masses to those expected.The coincidence rates of determination results of HA contents in seven batches of vaccine bulks by the alternative method and traditional SRID method were 87.90% ~ 122.20%.Conclusion An alternative method for determination of HA content in influenza vaccine is preliminarily developed,which may be used for the quantitative determination of HA in vaccine bulk when the WHO reference materials are not available in outbreak of pandemic influenza.