流感疫苗神经氨酸酶含量双抗体夹心ELISA定量检测方法的建立及应用

Development and application of a double antibody sandwich ELISA for neuraminidase content of influenza vaccine

目的建立用于定量检测流感疫苗神经氨酸酶(neuraminidase,NA)含量的双抗体夹心ELISA方法,并进行验证。方法采用多肽合成方式合成NA保守序列,经皮下多点免疫日本大耳白兔和豚鼠,共免疫5次,末次免疫后2周分别经颈动脉和心脏采血,分离血清,通过Protein G/A层析纯化,制备NA通用抗体。以鼠源通用抗体作为包被抗体,兔源通用抗体经HRP标记后作为酶标抗体,建立双抗体夹心ELISA法。确定包被抗体(32、16、8、4、2、1μg/mL)的工作浓度及酶标抗体(1∶50~1∶6400)的稀释度。验证方法的线性范围、准确度、重复性、中间精密度及耐用性。采用建立的方法测定流感裂解疫苗H1N1、H3N2、BV和BY型单价原液中的NA含量,并与荧光底物法进行比较。结果兔源和鼠源通用抗体的效价分别为128000和64000,纯度分别为95%和96%,蛋白浓度分别为2339和1780μg/mL。建立双抗体夹心ELISA法的最佳包被抗体工作浓度为16μg/mL,最佳酶标抗体稀释度为1∶400。NA(H3N2型)参考品在20~640 ng/mL浓度范围内与A450呈良好线性关系,R^(2)均>0.99;500、200、50 ng/mL的NA(H3N2型)参考品的平均样品回收率分别为102.15%、100.89%、100.70%,重复6次检测结果的CV均<10%,2名实验员3次检测结果的CV均<15%;不同抗原反应时间及酶标二抗孵育时间的样品回收率为85.41%~103.81%。建立的双抗体夹心ELISA法检测流感裂解疫苗H1N1、H3N2、BV、BY型单价原液NA含量分别为8.06、13.20、6.93、6.18μg/mL,荧光底物法检测的NA活性分别为29833、36800、28907、25871 U/L,两者结果呈正相关(R^(2)=0.9792)。结论建立的NA含量双抗体夹心ELISA定量检测法具有良好的准确度、重复性、中间精密度和耐用性,可用于流感疫苗的检定及生产过程中对NA含量的质量控制。

Objective To develop and verify a double antibody sandwich ELISA method for quantitative determination of neuraminidase(NA)content in influenza vaccine.Methods The conserved sequence of NA was synthesized by peptide synthesis,with which Japanese white rabbits and guinea pigs were immunized s.c.in several sites for 5 times.Serum samples were collected 2 weeks after the last immunization and purified by protein G/A chromatography to prepare a universal antibody.A double antibody sandwich ELISA method was developed using the universal antibody from guinea pigs as coating antibody and that from rabbits,labeled with horseradish peroxidase(HRP),as enzyme labeled antibody.The working concentration(32,16,8,4,2 and 1μg/mL)of coating antibody and dilution(1∶50~1∶6400)of enzyme labeled antibody were optimized.The developed method was verified for linear range,accuracy,reproducibility,intermediate precision and durability.The NA contents in monovalent bulks of influenza virus split vaccine of types H1N1,H3N2,BV and BY were determined by the developed method,and the result was compared with that by fluorescent substrate assay.Results The titers of universal antibodies from rabbit and guinea pig were 128000 and64000,while the purities were 95%and 96%,and the protein concentrations were 2339 and 1780μg/mL,respectively.The optimal working concentration of coating antibody was 16μg/mL,while the optimal dilution of enzyme labeled antibody was 1∶400.The concentration of reference of NA(type H3N2)at a range of 20~640 ng/mL showed a good linear relationship to the A450value,and all the R^(2)values were more than 0.99.The average recoveries of NA(type H3N2)reference at concentrations of 500,200 and 50 ng/mL were 102.15%,100.89%and 100.70%respectively.The CVs of six repeat test results were less than 10%,while those of three repeat tests by either of two workers were less than 15%.The recovery rates of samples when the antigen and enzyme labeled antibody were incubated for various time durations were 85.41%~103.81%.The NA contents of monovalent bulks of types H1N1,H3N2,BV and BY were 8.06,13.20,6.93 and 6.18μg/mL respectively,which was positively related to the NA activities determined by fluorescent substrate assay(29833,36800,28907 and 25871 U/L)(R^(2)=0.9792).Conclusion A double antibody sandwich ELISA method for quantitative determination of NA content was successfully developed,which showed good accuracy,reproducibility,precision and durability and might be used for control tests on influenza vaccine and quality control of NA content in the production process.