流感疫苗神经氨酸酶活性荧光底物检测法的建立及验证

Development and verification of fluorescent substrate assay for neuraminidase activity in influenza vaccine

目的 建立用于检测流感疫苗神经氨酸酶(neuraminidase,NA)活性的荧光底物法,并进行验证。方法 以唾液酸类似物4-MUNANA作为酶底物,通过测定NA催化底物分解后产物的荧光强度来评价NA活性。选取基于MDCK细胞制备的四价流感疫苗各单价原液(H1N1、H3N2、BV和BY)作为样品,优化荧光底物法的底物酶浓度(10、20、30、40、50μmol/L)、作用时间(每2 min检测1次,至90 min)、缓冲液pH(4.5、5.5、6.5、7.5、8.5和9.5)及反应温度(4、25、37和45℃)。以已知活性的重组NA蛋白作为参考品,验证方法的线性范围、准确度及精密度。用优化的方法检测流感疫苗生产过程中的样品(基于MDCK细胞制备的H1N1、H3N2、BV、BY相应的病毒收获液、纯化液和原液)及以MDCK细胞基质和鸡胚基质制备的四价流感裂解疫苗成品各1批。结果 最佳反应条件为:酶底物浓度20μmol/L,反应时间40 min,pH 7.5,反应温度37℃。重组NA蛋白参考品浓度在62.5~2 000 U/L范围内与荧光强度呈良好的线性关系,R2> 0.99;1 600、800、400 U/L 3个浓度参考品的回收率分别为92.60%~106.96%、85.54%~113.11%、87.00%~114.54%,于2个时间点共12次检测结果的CV分别为6.40%、8.25%、6.92%。H1N1、H3N2、BV和BY 4种亚型的流感疫苗半成品在不同阶段NA活性逐渐降低;细胞基质及鸡胚基质四价流感裂解疫苗成品的NA活性分别为61 585.82和69 583.51 U/L。结论 成功建立并优化了一种NA活性的荧光底物检测方法,该方法具有良好的准确度和精密度,可应用于流感疫苗生产过程及成品中NA活性的检测。

Objective To develop and verify a fluorescent substrate assay for neuraminidase(NA) activity in influenza vaccine. Methods Using 4-MUNANA[2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid],an analogues of salivary acid,as an enzyme substrate,the fluorescent strength of product after decomposition of NA catalytic substrate was determined to evaluate the activity of NA. Using the monovalent bulks of tetravalent influenza vaccine(H1N1,H3N2,BV and BY)prepared with MDCK cell substrate as samples,the substrate enzyme concentration(10,20,30,40 and 50 μmol/L),reaction time(the fluorescent strength was determined every 2 min for 90 min),pH value of buffer(4. 5,5. 5,6. 5,7. 5,8. 5 and 9. 5)and reaction temperatures(4,25,37 and 45 ℃)were optimized. The developed method was verified for linear range,accuracy and precision serving the recombinant NA protein with known activity as a reference. The sample during production of influenza vaccine(the corresponding virus harvests,purified virus liquids and bulks to H1N1,H3N2,BV and BY) as well as final products prepared with MDCK cell and chick embryo substrates,one batch for each,were determined by the optimized method. Results The optimal concentration of enzyme substrates,reaction time,pH value and reaction temperature were 20 μmol/L,40 min,7. 5 and 37 ℃ respectively. The recombinant NA concentration at a the range of 62. 5 ~ 2 000 U/L showed good linear relationship to the fluorescent strength,with a R2value of more than 0. 99.The recovery rates of references at concentrations of 1 600,800 and 400 U/L were 92. 60% ~ 106. 96%,85. 54% ~113. 11% and 87. 00% ~ 114. 54%,while the variation coefficients(CVs)of 12 determination results at two time points were 6. 40%,8. 25% and 6. 92%,respectively. The NA activities of final bulks of subtypes H1N1,H3N2,BV and BY sample decreased gradually at various stages. The NA activities of final products of tetravalent influenza split vaccine prepared with cell and chicken embryo substrates were 61 585. 82 and 69 583. 51 U/L respectively. Conclusion A fluorescent substrate assay for NA activity in influenza vaccine was developed,which showed high accuracy and precision and might be used for determination of NA activity of influenza vaccine during production and in final product.