Fluorogenic enzyme-linked immunosorbent assay with a dual color variation

The development of accurate and high-throughput biomarker detection tools is crucial for the diagnosis, monitoring, and treatment of various diseases. In this study, a sensitive fluorogenic enzyme-linked immunosorbent assay (FELISA) using Amplex Red or QuantaBlu fluorescent substrate was developed for the detection of tumor necrosis factor alpha and programmed cell death-ligand 1. The limit of detection of FELISA was in the nanogram order and multiple samples were conveniently assayed within 20 h using FELISA, demonstrating its applicability as a powerful immunoassay tool. FELISA can be widely used for rapid and accurate TNFα and PDL1 detection and applied to various fluorogenic immunoassays against other antigens of interest.