流感病毒神经氨酸酶抑制活性测定方法的标准研究

A study on the standard of influenza neuraminidase inhibition assay

建立流感病毒神经氨酸酶(NA)活性测定和酶抑制剂评价标准方法,并评价其准确性、稳定性。确定标准方法为:在96孔板内加入样品Vsam=10μL、酶溶液VNA=30μL、底物Vsub=60μL,混匀,37℃反应1 h,用NaOH终止反应,测定荧光强度。定义该反应条件下1 h内反应生成1 nmol 4-MU的NA酶量为一个活性单位。对测定结果,引入不确定度考察其准确性和稳定性。酶活性抑制率的合成相对不确定度为6.51×10?2,连续10天测得的活性化合物奥司米韦酸抑制活性结果稳定。本研究系统评价了NA抑制活性测定方法,为其在新药发现中的应用提供了实验依据,并探讨了对生物学指标测定的不确定度评价。

In present study,standard method and standard operation practice for measuring the activities of influenza neuraminidase and its inhibitors have been established.The accuracy and stability of the method has been evaluated.Standard operation is as following： 10 μL sample,30 μL neuraminidase and 60 μL substrate are added to one well of a 96-well plate,and then incubated at 37 ℃ for 1 h.The reaction was stopped with NaOH before fluorescence intensity determination.One unit of neuraminidase is defined as the amount of enzyme that produces 1 nmol 4-MU in 1 h under above conditions.The inhibition accuracy is indicated by an uncertainty measurement of 6.51×10-2,and its stability was reaffirmed by determination of oseltamivir acid.In this study,systematic assessment of neuraminidase inhibitory assay not only provided theoretical basis of its application in drug discovery,but also made preliminary attempt to use uncertainty measurement as a parameter in biological measurement.