BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.