甲型H1N1流感疫苗中神经氨酸酶含量双抗体夹心ELISA检测方法的建立与应用

Development and application of a double antibody sandwich ELISA for detection of neuraminidase contents in 2009 pandemic H1N1 influenza vaccine

目的建立甲型H1N1流感疫苗中神经氨酸酶（Neuraminidase,NA）含量双抗体夹心ELISA检测方法,并对其进行验证及初步应用。方法以HCA3通用抗体作为包被抗体,N1抗血清作为显示抗体建立双抗体夹心ELISA,确定线性范围,验证该方法的准确度及精密度,并用该方法对3个厂家共9批次甲型H1N1流感疫苗中的NA含量进行测定。结果 NA质量浓度在0~50 ng/m L时线性良好（r2〉0.99）;回收率为97.54%~104.02%;实验内CV〈10%,实验间CV〈15%。不同厂家的甲型H1N1流感疫苗中NA含量差异很大,NA与HA（血凝素）的百分比最高达到29.85%,而最低的只有7.00%;而同一厂家各批次疫苗间的NA与HA的比例较为稳定。结论成功建立了甲型H1N1流感疫苗中NA含量双抗体夹心ELISA检测方法,该法具有良好的线性及灵敏度,准确度和精密度高,能满足甲型H1N1流感疫苗NA含量的检测需求。

Objective To develop a double antibody sandwich ELISA for detection of neuraminidase（NA） contents in pandemic H1N1 influenza vaccine,and its verification and preliminary application was performed. Methods A double antibody sandwich ELISA was set up by using an universal NA antibody HCA3 as capture antibody and N1 antiserum as a signal antibody. The recovery rate and coefficients of variation in intra-assay and inter-assay were calculated to evaluate the linear range and precision / accuracy of this method. NA contents were quantified in 9 lots 2009 pandemic H1N1 influenza vaccine from 3companies. Results The ELISA showed a good linearity when NA concentration was in a range from 0 to 50 ng / m L with r2〉0.99,the recovery rates were from 97.54% to 104.02%. Coefficients of variation within intra assay was below 10% but among inter assay was below 15%. The NA contents were quite different in the vaccines from different companies. The hightest percentage of NA to HA（ Hemmagglutin） reached 29.85%,on the other hand,the lowest only 7.00%,and moreover,the ratio of NA to HA was more stable among the vaccine batches from the same company. C onclusion The double antibody sandwich ELISA was successfully set up and the method with a good linearity and sensitivity,high accuracy and precision,by which qualification NA content could be performed in pandemic H1N1 influenza vaccine.