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.