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

Abstract 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.