Plate-Adherent Nanosubstrate for Improved ELISA of Small Molecules: A Proof of Concept Study

Enzyme-linked immunosorbent assay (ELISA) is a widely used technique for detecting and quantifying target analytes in clinical and research laboratories. One of the main drawbacks of ELISA is the involvement of multiple washing steps that desorbs the capture antigen/antibody off the polystyrene plate, thereby producing inconsistent and erroneous data. To overcome the problem of desorption, we hypothesized that gelatin nanoparticles (GelNP) could serve as a "plate-adherent" substrate to irreversibly adhere the capture antigen/antibody of interest. We tested our hypothesis using GelNP-based substrate (Gel–BSA–OHG) to adhere 8-hydroxy-2′-deoxyguanosine (8-OHdG) to the polystyrene plate and assayed this molecule using the ELISA technique. The stability and ELISA performance of Gel–BSA–OHG was evaluated in comparison to the conventional substrate (BSA–OHG). Importantly, the Gel–BSA–OHG substrate was found to be more wash-resistant and consequently resulted in improved sensitivity, accuracy, and precision in the ELISA analysis of 8-OHdG. Finally, the scope of Gel–BSA–OHG substrate-based ELISA for clinical application was demonstrated by validating its ability to detect 8-OHdG in an artificial urine sample with high specificity.