一种氨肽酶N抑制剂抑酶活性检测试剂盒的开发

Development of detection kit of inhibitory activity of aminopeptidase N inhibitor

目的开发一种能准确、特异地评价目标化合物对人氨肽酶N的抑酶活性检测试剂盒。方法验证试剂盒准确度、精密度、稳定性等性能指标,同时通过与同类的传统试剂盒进行米氏常数的评价确认该试剂盒的灵敏度。结果精密度验证,试剂盒的CV=4.29%符合CV<15%的要求;准确度验证中,标准化残差δ^(*)=1.747落在(-2,2)区间以内,在95%置信度内是正常实验点,数据准确可靠;酶源稳定性良好;线性范围宽,96孔板中氨肽酶N在加入量120 μL时还能呈现良好的线性关系;该试剂盒米氏常数低于传统试剂盒,说明灵敏度高。结论所开发的试剂盒主要的性能指标表现良好,能准确测定化合物对人源氨肽酶N的抑酶活性,其检测成本和可靠性优于现有上市产品,具有推广应用及开发价值。

Objective To develop a kit to accurately evaluate the inhibitory effect of target compounds against human aminopeptidase N(APN).Methods The accuracy,precision,stability and other performance indicators of the kit were verified and the sensitivity of the kit was confirmed through evaluating the Km with similar traditional kit.Results According to the precision verification,CV=4.29%of this kit met the requirement of CV<15%.In the accuracy verification,the standardized residual δ^(*)=1.747 fell within the(-2,2)interval,which was a normal experimental point within 95%confidence.Therefore,the data were accurate and reliable.The enzyme source showed good stability.The linear range was wide.A good linearity was shown when 120μL APN enzyme was added into the 96-well plate.Km of this kit was lower than the traditional kit,indicating high sensitivity.Conclusion The main indicators of this kit perform well,which can accurately determine the inhibitory activity of the compound against human APN.The cost and reliability of the test are superior to the market products.