Protease-associated enzyme-linked immunosorbent assay for detecting matrix metalloproteinase-9

Matrix metalloproteinase-9 (MMP-9) plays a key role in extracellular matrix remodeling and is implicated in various physiological and pathological processes, including tissue regeneration and cancer progression. Accurate quantification of MMP-9 is essential for diagnostic and therapeutic applications. In this study, we developed a protease-based fluorescence enzyme-linked immunosorbent assay (ELISA) platform utilizing biotinylated α-chymotrypsin (α-CTB) as an alternative enzymatic reporter to conventional horseradish peroxidase (HRP). α-CT was conjugated with biotin using biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester, achieving a labeling ratio of approximately 6.5:1 without significantly compromising enzymatic activity. The biotinylation efficiency was verified using the 2-(4-hydroxyphenylazo)benzoic acid (HABA)–avidin assay, and proteolytic activity was assessed with the fluorogenic substrate. Comparative kinetic analysis revealed that α-CTB retained substantial activity, including when immobilized on streptavidin-coated surfaces, validating its use in solid-phase immunoassays. The α-CTB-based ELISA was applied to quantify MMP-9 and compared with a conventional HRP-based ELISA. The α-CTB assay exhibited a concentration-dependent increase in fluorescence across a broad dynamic range, achieving a lower limit of quantification (LOQ) of 1.49 ng/mL, whereas the traditional ELISA showed reduced sensitivity at low concentrations with an LOQ of 3.19 ng/mL. This improved sensitivity demonstrates the suitability of α-CTB as an alternative enzymatic reporter for accurate biomarker quantification.