[A study on the standard of influenza neuraminidase inhibition assay].
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 microL sample, 30 microL neuraminidase and 60 microL substrate are added to one well of a 96-well plate, and then incubated at 37 degrees C 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 x 10(-12), 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.
Beijing Key Laboratory of Drug Target and Screening Research, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
Fan
F
Yang
Ai-Lin
AL
Liu
Guan-Hua
GH
Du
D000998:Antiviral Agents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D053122:Influenza A Virus, H3N2 Subtype / Q000201:enzymology*; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D053139:Oseltamivir / Q000494:pharmacology*
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D053139:Oseltamivir; D006923:Hymecromone; D009439:Neuraminidase