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目的优化发色底物法,使其在酶促反应初速度内测定α1抗胰蛋白酶(AAT)的活性并用于血浆蛋白纯化过程中各样品活性的检测。方法采用酶标仪动态监测模式观察酶浓度和反应时间对酶促反应的影响;计算初速度并确定被底物饱和的最大酶浓度。将AAT与胰蛋白酶孵育,剩余靶酶和底物作用产生的光密度可反映AAT的活性。通过△D与AAT标准品活性进行拟合建立标准曲线,测定相关样品的活性,进行精密度和准确度验证。结果胰蛋白酶浓度为0.0625 mg/ml,20 min内酶促反应处于初速度内。AAT标准曲线范围为200~1200 IU/ml,r>0.99。该法测定CohnⅣ、前处理液、洗脱峰样品中AAT的活性分别为(720.59±18.63)、(601.84±19.18)、(568.09±24.83)IU/ml。每个样品之间的RSD<10%,加样回收率均在90%~110%。结论发色底物法经优化后,准确度和精密度大幅度提高,更适于实验室制备AAT或不同生产阶段样品的活性检测。
OBJECTIVE: To optimize the chromogenic substrate method to measure the activity of α1 antitrypsin (AAT) in the initial velocity of enzymatic reaction and to detect the activity of each sample in the process of plasma protein purification. Methods The effect of enzyme concentration and reaction time on the enzymatic reaction was observed by the dynamic monitoring mode of microplate reader. The initial velocity was calculated and the maximum enzyme concentration saturated by the substrate was determined. The AAT and trypsin incubation, the remaining target enzyme and the substrate produced optical density can reflect the activity of AAT. Through the △ D and AAT standard activity fitting to establish a standard curve to determine the activity of the relevant samples for precision and accuracy verification. Results The trypsin concentration was 0.0625 mg / ml and the enzymatic reaction was within the initial velocity within 20 min. AAT standard curve range of 200 ~ 1200 IU / ml, r> 0.99. The activity of AAT in Cohn IV, pretreatment solution and elution peak was (720.59 ± 18.63), (601.84 ± 19.18) and (568.09 ± 24.83) IU / ml, respectively. The RSD between each sample was <10% and the sample recovery was between 90% and 110%. Conclusion The chromogenic substrate method has been greatly optimized in accuracy and precision, and is more suitable for the laboratory test of AAT or different samples in different production stages.