Development and validation of novel diagnostic recombinant surface antigen D5 and cytotoxic gene pathogenesis island Cag11 protein for Helicobacter pylori

Helicobacter pylori, a highly diverse bacterium, poses a significant health threat globally, causing a range of chronic gastritis to potentially fatal gastric cancer. The World Health Organization (WHO) has advocated for an Accurate Test-and-Treat Strategy (ATTS) to combat this infection. Despite advancements in diagnostic assays for H. pylori, the existing biomarkers face limitations due to the bacterium's antigenic variations across different populations. This variability significantly impacts the sensitivity and specificity of diagnostic tests, particularly when strain origin and serum sample origins differ. Furthermore, lack of a standard test for diagnosing H. pylori, combined with limited diagnostic tools and genetic variations in regions like Africa, makes it difficult to accurately diagnose and measure the prevalence of H. pylori infection. To overcome these challenges, researchers have focused on developing novel, non-invasive diagnostic assays using highly specific, conserved and immunogenic biomarkers. Thus, this study investigated the potential of two proteins, PSA D15 and Cag11, as diagnostic targets. Through in-silico analysis, these proteins were identified as having excellent characteristics for diagnostic applications, including high stability, solubility, and immunogenicity. To further validate these findings, the designed antigens were synthesized, expressed recombinant versions of PSA D15 and Cag11 (rPSA D15 and rCag11) in E. coli BL21(DE3) and purified using Ni-NTA affinity chromatography. Subsequently, the purified ~ 24 kDa rPSA D1 and ~ 21 kDa rCag11 proteins were confirmed by SDS-PAGE and Western blotting. Evaluation of the diagnostic performance of the newly developed rPSA D15-ELISA assay revealed optimum cut-off value of 2.246% with 93.33% (95% CI 78.68-98.82) sensitivity, 96.67% (95% CI 83.33-98.83) of specificity, AUC of 0.92 (95% CI  0.838-1.000) and P < 0.0001. Similarly, the rCag11-ELISA assay also showed optimum cut-off value of 2.346% with 96.67% (95% CI 83.33-99.83) sensitivity, 96.67% (95% CI 83.33-99.83) specificity, AUC of 0.99 (95% CI   0.98-1.000) and P value < 0.0001. Furthermore, the coefficient of variation (%CV) of reproducibility assays of the newly developed rPSA D15-ELISA was less than 10%, indicating that the two ELISA assays exhibit excellent reproducibility, reliability and could be used for routine detection. The comparative study results of rPSA D15-ELISA and rCag11-ELISA showed a high agreement (k = 0.767 and 0.833) with the commercially available H. pylori antibody test immunochromatographic kit. Overall, the results suggest that the newly developed rPSA D15 and rCag11 antigens represent promising diagnostic biomarkers for accurate and reliable detection of H. pylori infection, particularly in African setting and globally in large.