发色底物法在酶促反应初速度内测定α1抗胰蛋白酶的活性  

Detection of α1 antitrypsin activity by chromogenic substrate assay with initial velocity of enzymatic reaction

作　　者：张晋超[1] 赵雄[1] 吕茂民[1] 尹惠琼[1] 王延琳[1] 章金刚[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《军事医学》2015年第3期189-192,共4页Military Medical Sciences

基　　金：国家863计划资助项目(2012AA021902)

摘　　要：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 detect the activity of cd antitrypsin（AAT） with initial velocity of enzymatic reaction in order to detect the activity of samples in the process of separating and purifying plasma protein, chromogenic substrate assay was optimized. Methods The effect of trypsin concentration and reaction time on enzymatic reaction was acquired by the kinetic monitoring mode of the microplate reader. Initial velocity was calculated to confirm the largest concentration of trypsin which was saturated by substrate. AAT was incubated with trypsin and absorbanee produced by enzymatic reaction of remaining trypsin and substrate could reflect the activity of AAT. A standard curve was established with △D fitting with the activity of AAT standard. The activity of related samples was detected and the precision and accuracy of the method were evaluated. Results Trypsin concentration was 0. 0625 mg/ml. Within 20 minutes, enzymatic reaction was with initial velocity. The range of the standard curve was 200 - 1200 IU/ml. Correlation coefficient was more than 0.99. The activity of Cohn IV, samples of pre-processing and elution were （720.59 ± 18.63 ）, （601.84 ± 19.18）, and （568.09 ± 24.83 ）IU/ml, respectively. The relative standard deviation was less than 10%. Sample recovery rate was 90%- 110%. Conclusion The optimized chromogenic substrate assay greatly improves accuracy and precision. The method can be used for the detec- tion of AAT activity of samples in laboratories and workshops.

关 键 词：发色底物法 酶促反应初速度 Α1抗胰蛋白酶 活性测定 

分 类 号：R392-33[医药卫生—免疫学]