Chemiluminescent ELISA based on enzyme-mediated hydrogen peroxide supply

Enzyme-linked immunosorbent assays (ELISA) commonly use horseradish peroxidase (HRP) to detect chromogenic and chemiluminescent signals. The enzymatic oxidation of these probes requires hydrogen peroxide, which is inherently unstable at ambient temperature and pH, thus affecting the reproducibility and long-term storage stability of the assays. In this study, an enzyme-mediated peroxide supply using glucose oxidase (GOD)/glucose was established as an alternative source of hydrogen peroxide for chemiluminescent ELISA. The performance of the system was evaluated relative to that of conventional ELISA using hydrogen peroxide solution. The optimal reaction conditions were estimated, including GOD and glucose concentrations, as well as the reaction time for the chemiluminescent ELISA. The performance of the enzyme-mediated hydrogen peroxide supply was demonstrated using four commercial ELISA kits for the detection of alpha-fetoprotein (AFP), hepatitis B surface antigen (HBsAg), human immunodeficiency virus 1/2 antibody (HIV-1/2 Ab), and carcinoembryonic antigen (CEA). The GOD-mediated hydrogen peroxide supply-based ELISA achieved a limit of detection (LOD) comparable to that obtained with the conventional hydrogen peroxide condition, a high statistical agreement (|mean bias| ≤ 0.02, |95% limit of agreement (LoA)| ≤ 0.15, Spearman's ρ > 0.96).