{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T03:21:13Z","timestamp":1782789673175,"version":"3.54.5"},"reference-count":28,"publisher":"Public Library of Science (PLoS)","issue":"1","license":[{"start":{"date-parts":[[2025,1,15]],"date-time":"2025-01-15T00:00:00Z","timestamp":1736899200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p><jats:italic>H<\/jats:italic>. <jats:italic>pylori (Hp)<\/jats:italic> is highly causative agent of chronic gastritis, gastric cancer and human death worldwide. To address the challenge of <jats:italic>H<\/jats:italic>. <jats:italic>pylori<\/jats:italic> infection, numerous immunological assays have been developed for its diagnosis and management. However, the limited availability of these assays in certain laboratories, coupled with their high cost, inconsistent specificity, and sensitivity, has hampered their widespread adoption, particularly in developing countries where <jats:italic>H<\/jats:italic>. <jats:italic>pylori<\/jats:italic> infection is prevalent. Therefore, this study aimed to develop and validate a competitive enzyme-linked immunosorbent assay (cELISA) assay for detecting <jats:italic>H<\/jats:italic>. <jats:italic>pylori<\/jats:italic> infections by targeting the Protective Surface Antigen (PSA) and Cytotoxic-Associated Gene Pathogenesis Island (Cag11) proteins in <jats:italic>H<\/jats:italic>. <jats:italic>pylori<\/jats:italic> stool antigen sample. In the current study, the optimal conditions including the dilution of anti-rPSA D15 and anti-rCag11 antibodies at 1:1000, coating antigens (rPSA D15 and rCag11) at a concentration of 1 \u03bcg\/well, the dilution of HRP-labelled antibody at 1:5000 and <jats:italic>H<\/jats:italic>. <jats:italic>pylori<\/jats:italic> stool antigen dilution at 1:1000 with a 1hour incubation and color development time of 30 minutes for cELISA were determined using an ELISA checkerboard titration assay. Based on the optimized conditions, novel rPSA D15-cELISA and rCag11-cELISA assays with a respective optimum cut-off value of 20.80% PI and 24.16% PI were developed. According to the receiver operating characteristic (ROC) curve analysis on the diagnostic performance of the newly developed rPSA D15-cELISA and rCag11-cELISA assays using 60 clinical <jats:italic>H<\/jats:italic>. <jats:italic>pylori<\/jats:italic> stool samples, the rPSA D15-cELISA test assay established an optimum cut-off point of 20.80% with sensitivity and specificity of 90% (95% confidence of interval (CI) 74.38\u201396.54), Area under the curve (AUC) of 0.9556 (95% CI = 0.896\u20131.000) and P value &lt;0.0001. Similarly, the rCag11-cELISA assay revealed optimum cut-off value of 24.16% with sensitivity of 93.33% (95% CI 78.68\u201398.82), specificity of 90% (95% CI 74.38\u201396.54), AUC of 0.986 (95% CI = 0.967\u20131.000) and P &lt;0.0001. Furthermore, the reproducibility assay coefficients of variation (CV) of the newly developed rPSA D15-cELISA and rCag11-cELISA assay were less than 10%, indicating that the two cELISA assays exhibits excellent reproducibility and reliability. To validate their clinical diagnostic application, the comparative study results of rPSA D15-cELISA and rCag11-cELISA showed a high agreement (k = 0.766 and 0.799) with the commercially available <jats:italic>H<\/jats:italic>. <jats:italic>pylori<\/jats:italic> antigen test immunochromatographic kit and more accurate than the reference kit by detecting stool antigen of <jats:italic>H<\/jats:italic>. <jats:italic>pylori<\/jats:italic> strain, indicating it is promising for clinical testing. In conclusion, these results indicated that the newly developed rPSA D15-cELISA and rCag11-cELISA <jats:italic>H<\/jats:italic>. <jats:italic>pylori<\/jats:italic> stool antigen test assays were a potential reliable and a clinically useful assay for rapid, specifically, sensitively and accurately diagnosis and large-scale epidemiological investigation of <jats:italic>H<\/jats:italic>. <jats:italic>pylori<\/jats:italic> infection.<\/jats:p>","DOI":"10.1371\/journal.pone.0317227","type":"journal-article","created":{"date-parts":[[2025,1,15]],"date-time":"2025-01-15T18:33:27Z","timestamp":1736966007000},"page":"e0317227","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":4,"title":["Development and optimization of a new competitive ELISA using recombinant (rPSA D15 and rCag11) antigens for the detection of Helicobacter pylori infection"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3219-305X","authenticated-orcid":true,"given":"Biniam Moges","family":"Eskeziyaw","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naomi","family":"Maina","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebecca","family":"Waihenya","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew Mutinda","family":"Munyao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tonny Teya","family":"Nyandwaro","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shingo","family":"Inoue","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5789-4434","authenticated-orcid":true,"given":"Samson Muuo","family":"Nzou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,1,15]]},"reference":[{"issue":"3","key":"pone.0317227.ref001","doi-asserted-by":"crossref","DOI":"10.1016\/j.heliyon.2022.e09189","article-title":"Evaluation of the diagnostic performance and the utility of Helicobacter pylori stool antigen lateral immunochromatography assay","volume":"8","author":"S Abdelmalek","year":"2022","journal-title":"Heliyon"},{"issue":"36","key":"pone.0317227.ref002","doi-asserted-by":"crossref","first-page":"12781","DOI":"10.3748\/wjg.v20.i36.12781","article-title":"Beyond the stomach: an updated view of Helicobacter pylori pathogenesis, diagnosis, and treatment. 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