Roda A, Piazza F, Pasini P, Baraldini M, Zambonin L, Fossi S, Bazzoli F, Roda E
Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy.
Anal Biochem. 1998 Nov 1;264(1):47-52. doi: 10.1006/abio.1998.2811.
A chemiluminescent urease activity assay has been developed and optimized using the chemiluminescent pH indicator phthalhydrazidylazoacetylacetone. This compound is stable at pH </= 7 and decomposes at higher pH values, emitting light in the presence of H2O2. Urease catalyzes hydrolysis of urea to form NH3 and CO2 which increase the pH of the reaction medium, thus allowing the chemiluminescent indicator to decompose and produce photons. The emitted light is proportional to the urease activity when urea is in excess. Urease tests based on colorimetric pH indicators like phenol red are commercially available and commonly used for the rapid diagnosis of Helicobacter pylori infection in gastric mucosa biopsy specimens, since this bacterium produces high amounts of urease. Such colorimetric tests often lack sensitivity, giving false-negative results. The developed chemiluminescent test proved to be at least 50-fold more sensitive than the colorimetric tests, permitting early diagnosis of infection, and it is more rapid, giving results in 1-10 min compared to 30 min. Further applications of this assay could be the in situ localization of urease activity, corresponding to the presence of H. pylori, in gastric mucosa cryosections and the development of high-throughput screening assays of antimicrobial drugs able to inactivate the bacterium.
利用化学发光pH指示剂邻苯二甲酰肼基偶氮乙酰丙酮，开发并优化了一种化学发光脲酶活性测定方法。该化合物在pH≤7时稳定，在较高pH值下分解，在H2O2存在时发光。脲酶催化尿素水解形成NH3和CO2，这会增加反应介质的pH值，从而使化学发光指示剂分解并产生光子。当尿素过量时，发出的光与脲酶活性成正比。基于比色pH指示剂（如酚红）的脲酶检测试剂盒在市场上有售，常用于胃黏膜活检标本中幽门螺杆菌感染的快速诊断，因为这种细菌会产生大量脲酶。此类比色检测通常缺乏灵敏度，会出现假阴性结果。已开发的化学发光检测方法被证明比色检测至少灵敏50倍，能够实现感染的早期诊断，而且速度更快，1 - 10分钟即可得出结果，而比色检测需要30分钟。该检测方法的进一步应用可能包括在胃黏膜冷冻切片中对脲酶活性进行原位定位（这与幽门螺杆菌的存在相对应），以及开发能够使该细菌失活的抗菌药物高通量筛选检测方法。
