A Pilot Study to Develop an Ultrasensitive Thio-NAD Cycling ELISA for Neisseria gonorrhoeae Detection

Neisseria gonorrhoeae and its increasing antimicrobial resistance have raised global health concerns, creating an urgent need for more advanced diagnostics to contain the spread. We conducted a preclinical trial using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) with Thio-nicotinamide-adenine dinucleotide (NAD) cycling for the detection of N. gonorrhoeae. Three independent researchers conducted the initial experiments to obtain calibration curves. The inter-assay reproducibility across the 3 investigators yielded coefficients of variation of 11% when measuring 500000 CFU/mL, confirming the consistent performance of the ultrasensitive ELISA. Specificity against common clinical microorganisms was also assessed. Performance was first evaluated in simulated urine, vaginal fluid, and saliva. A comparative evaluation of 90 clinical isolates was then performed using a conventional ELISA method. The ultrasensitive ELISA produced consistent calibration curves (R² = 0.99) with limits of detection of 1.1 × 10⁴ to 1.8 × 10⁴ CFU/mL. No cross-reactivity to common pathogens was observed. The assay results revealed linearity in simulated fluids, with a modestly reduced sensitivity in urine and vaginal fluid. At 50000 CFU/mL, the ultrasensitive ELISA generated signals >50-fold stronger than the p-nitrophenyl phosphate (pNPP) ELISA. For clinical isolates, the ultrasensitive ELISA identified 78.9% as positive compared with 30.0% by pNPP ELISA (p = 0.0005). The ultrasensitive ELISA detected N. gonorrhoea with high sensitivity and specificity across sample types, consistently outperforming the conventional ELISA, and demonstrating the potential of this approach as a cost-effective diagnostic alternative.