Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Helicobacter. 2011 Feb;16(1):42-6. doi: 10.1111/j.1523-5378.2010.00813.x.
Nowadays, there is an increasing interest in noninvasive methods to diagnose Helicobacter pylori infection. Indeed, they can profitably replace endoscopy in predicting the diagnosis. The stool antigen test for H. pylori is a noninvasive immunoassay to diagnose active infection with this bacterium in human fecal samples. The aim of this study was detection of alkyl hydroperoxide reductase protein (AhpC) antigen by immunoblotting in stool samples for diagnosis of H. pylori.
Chromosomal DNA from H. pylori was isolated. AhpC gene was amplified by PCR, These amplicons were cloned into pTZ57R/T cloning vector then subcloned into pQE30 expression vector and overexpressed using isopropyl-beta-D-thiogalactopyranoside in E. coli M15. AhpC protein was purified by affinity chromatography. Rabbits were immunized with the purified AhpC protein for the production of antibodies. To determine the accuracy of the test for diagnosing H. pylori infection from stool, we evaluated 84 patients (6-81 years old) using Western blot analysis by rabbit anti-AhpC antibody. Positive rapid urease test on biopsy samples was considered as the gold standard.
AhpC gene was overexpressed, and AhpC protein was purified. Rabbit anti-AhpC antibody produced after immunization with the purified AhpC protein. By immunoblotting, we detected AhpC protein in the positive stool samples. The test showed a 83.3% sensitivity (95% CI: 69.8-92.5%) and a 91.7% specificity (95% CI: 77.5-98.2). Among the children, the sensitivity was 88.2% (95% CI: 63.6-98.5) and the specificity was 100% (95% CI: 69.2-100); in adults, the sensitivity and specificity were 80.6% (95% CI: 62.5-92.5) and 88.5% (95% CI: 69.8-97.6), respectively.
Using of AhpC antigen for diagnosis of H. pylori infection is a useful noninvasive method, accurate in adolescents and children, and can be used for the development of a stool antigen detection kit for H. pylori.
如今，人们对非侵入性方法诊断幽门螺杆菌感染越来越感兴趣。事实上，这些方法可以在预测诊断方面替代内窥镜检查。幽门螺杆菌粪便抗原检测是一种非侵入性免疫测定法，用于检测人类粪便样本中这种细菌的活性感染。本研究的目的是通过免疫印迹法检测粪便样本中的烷基氢过氧化物还原酶蛋白 (AhpC) 抗原，以诊断幽门螺杆菌。
从幽门螺杆菌中分离出染色体 DNA。通过 PCR 扩增 AhpC 基因，将这些扩增子克隆到 pTZ57R/T 克隆载体中，然后亚克隆到 pQE30 表达载体中，并用异丙基-β-D-硫代半乳糖苷在大肠杆菌 M15 中过表达。通过亲和层析纯化 AhpC 蛋白。用纯化的 AhpC 蛋白免疫兔子以产生抗体。为了确定从粪便中诊断幽门螺杆菌感染的检测准确性，我们使用 Western blot 分析评估了 84 名患者（6-81 岁），使用兔抗 AhpC 抗体。将活检样本中快速尿素酶检测阳性作为金标准。
AhpC 基因过表达，AhpC 蛋白得到纯化。用纯化的 AhpC 蛋白免疫兔子后产生兔抗 AhpC 抗体。通过免疫印迹，我们在阳性粪便样本中检测到 AhpC 蛋白。该检测的敏感性为 83.3%（95%CI：69.8-92.5%），特异性为 91.7%（95%CI：77.5-98.2%）。在儿童中，敏感性为 88.2%（95%CI：63.6-98.5%），特异性为 100%（95%CI：69.2-100%）；在成人中，敏感性和特异性分别为 80.6%（95%CI：62.5-92.5%）和 88.5%（95%CI：69.8-97.6%）。
使用 AhpC 抗原诊断幽门螺杆菌感染是一种有用的非侵入性方法，在青少年和儿童中准确，可用于开发幽门螺杆菌粪便抗原检测试剂盒。
