Detection of proteases using an immunochemical method with haptenylated-gelatin as a solid-phase substrate
Detection of proteases using an immunochemical method with haptenylated-gelatin as a solid-phase substrate
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DOI：
10.1007/s00216-010-3540-z
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发表时间：
2010-04-01
影响因子：
4.3
通讯作者：
Price, Robert G.
中科院分区：
文献类型：
--
作者：
Abuknesha, Ramadan A.;Jeganathan, Fiona;Price, Robert G.
描述了一种利用结合ELISA终点检测步骤的固相底物测量蛋白酶的简化方法。发现吸附在聚苯乙烯表面上的明胶半抗原缀合物是蛋白酶的有效底物。固相蛋白质-半抗原复合物的消化导致所连接的缀合物的成比例解吸和可检测的半抗原种类的减少。明胶-胆酸结合物，亲和纯化羊抗胆酸抗体-HRP和显色底物被纳入一个方便和高度敏感的固相免疫化学方法。可检测信号与酶活性成反比。测定了细菌蛋白酶（α-糜蛋白酶II型、来自多粘芽孢杆菌的IX型、来自灰色链霉菌的XIV型、来自地衣芽孢杆菌的XXIV型）。在0-550 μ单位mL（-1）（胰凝乳蛋白酶）、0-12 μ单位mL（-1）（IX型）、0-35 μ单位mL（-1）（XIV型）和0100 μ单位mL（-1）（XXIV型）范围内测量酶活性的剂量-反应曲线。所研究的蛋白酶的检测极限为胰凝乳蛋白酶89 μ单位mL（-1），IX型0.26 μ单位mL（-1），XIV型5.8 μ单位mL（-1）和XXIV型6.5 μ单位mL（-1）。事实证明，两步免疫化学方法结合了固相酶免疫测定的简单性和灵敏度、明胶作为蛋白酶底物的广泛特异性以及固相形式的灵活性。
A simplified method for the measurement of proteases utilising solid-phase substrates incorporating an ELISA end-point detection step is described. Gelatinhapten conjugates adsorbed onto polystyrene surfaces were found to be efficient substrates for proteases. Digestion of the solid-phase protein-hapten complexes resulted in proportional desorption of the attached conjugates and decrease in the detectable hapten species. Gelatin-cholic acid conjugates, affinity-purified sheep anti-cholic acid antibody-HRP and a chromogenic substrate were incorporated into a convenient and highly sensitive solid-phase immunochemical method. The detectable signal is inversely proportional to enzyme activity. Bacterial proteases (alpha-chymotrypsin Type II, Type IX from Bacillus polymyxa, Type XIV from Streptomyces griseus, Type XXIV from Bacillus licheniformens) were assayed. Dose-response curves for enzyme activities were measured within ranges of 0-550 mu units mL(-1) for chymotrypsin, 0-12 mu units mL(-1) for type IX, 0-35 mu units mL(-1) for type XIV and 0100 mu units mL(-1) for type XXIV. The detection limits of the proteases studied were 89 mu units mL(-1) for chymotrypsin, 0.26 mu units mL(-1) for type IX, 5.8 mu units mL(-1) for type XIV and 6.5 mu units mL(-1) for type XXIV. It was demonstrated that the two-step immunochemical method combines the simplicity and sensitivity of solid-phase enzyme immunoassays, the broad specificity of gelatin as a protease substrate and the flexibility of the solid-phase format.