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目的建立微量滴定板上的弹性蛋白酶动态显色法(以下简称微量滴定板法),用于α1-蛋白酶抑制剂(α1-Pro-teinase inhibitor,α1-PI)的活性检测。方法以微量滴定板为载体,将不同量的α1-PI样品与定量的弹性蛋白酶混合,通过适宜的显色底物对剩余的弹性蛋白酶活性进行测定。用酶标仪测定405 nm处A值的变化,分析试验的有效性,并计算α1-PI样品的活性。优化微量滴定板法的实验条件。用BCS凝集分析仪检测α1-PI的活性,确认样品的工作浓度。采用两种方法检测19份样品的α1-PI活性,分析二者的相关性。结果微量滴定板法的最优实验条件为:样品加样体积50μl,标准曲线范围为(2～14)μg/ml;弹性蛋白酶稀释度为1∶1 400,体积100μl;孵育时间15 min;底物稀释度为1∶10,体积50μl;连续监测A405值10 min。微量滴定板法和BCS法检测19份样品的相关系数大于0.99。结论微量滴定板法检测α1-PI活性的时间更短,同一样品不同稀释度之间检测结果的变异率更低,且检测结果与BCS法相关性良好,可替代全自动的BCS法,降低检测成本。
OBJECTIVE To establish a dynamic colorimetric method for elastase on microtiter plates (hereinafter referred to as microtiter plate method) for the detection of the activity of α1-Pro-teinase inhibitor (α1-PI). Methods Using microtiter plates as carrier, different amounts of α1-PI samples were mixed with quantitative elastase, and the remaining elastase activity was determined by appropriate chromogenic substrate. The change of A value at 405 nm was measured with a microplate reader, the validity of the test was analyzed, and the activity of the α1-PI sample was calculated. Optimize the experimental conditions of microtiter plate method. The activity of α1-PI was measured by BCS agglutination analyzer to confirm the working concentration of the sample. Two methods were used to detect the activity of α1-PI in 19 samples, and their correlations were analyzed. Results The optimum conditions for the microtiter plate method were as follows: sample volume 50μl, standard curve range (2 ~ 14) μg / ml; elastase dilution 1:1 400, volume 100μl; incubation time 15 min; Material dilution 1:10, volume 50μl; continuous monitoring A405 value 10 min. The correlation coefficient of 19 samples tested by microtitre plate method and BCS method was more than 0.99. Conclusion The titration of α1-PI by microtitre plate method is shorter and the variation rate of detection results with different dilutions of the same sample is lower. Moreover, the test results have good correlation with BCS method, which can replace the automatic BCS method and reduce the detection cost.