High-affinity monoclonal antibody pairs-based time-resolved fluorescent microsphere-lateral flow and electrochemical immunosensing platforms for rapid and quantitative detection of Helicobacter pylori in saliva.

Current diagnostic methods for Helicobacter pylori (H. pylori) infection suffer from critical limitations. Endoscopy is invasive, while stool antigen tests and urea breath tests require specialized laboratories and complex sample processing. To address these unmet needs, we present a novel saliva-based point-of-care platform for rapid and quantitative H. pylori detection. The platform consists of two innovative biosensors: a time-resolved fluorescent microsphere-lateral flow immunoassay strip (TRFM-LFIA) and an electrochemical immunosensor, specifically designed for UreB antigen detection in unprocessed saliva. A high-affinity monoclonal antibody pair (2-C2-F5/8-B5-E6) targeting non-overlapping UreB epitopes was developed through integrated recombinant antigen expression, hybridoma technology, and molecular docking. This antibody pair was applied to both biosensors using a double-antibody sandwich format, achieving high sensitivity (LOD: 91 pg/mL for TRFM-LFIA; 38.3 pg/mL for electrochemical immunosensor) and broad dynamic range (0.1-1000 ng/mL) without sample pre-treatment. Both biosensors demonstrated good specificity against common interferents, with reproducibility below 8% coefficient of variation, and 100% concordance with gold-standard diagnostics in 21 saliva samples (11 H. pylori-positive, 10 healthy controls). Crucially, this work enables direct saliva analysis, eliminating the need for invasive procedures and complex infrastructure, thereby offering a scalable and cost-effective solution for resource-limited settings.