Quantitative Assessment of Bony Fish Tropomyosin Using a Monoclonal Antibody Competitive ELISA

Bony fish is one of the big-nine allergenic foods in the US. This study established a monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of bony fish tropomyosin (TM), a fish allergen. Immunoassay (Western blot and ELISA) was performed to characterize anti-TM mAb8F5 (target configuration, immunoaffinity, and species selectivity). Atlantic cod (<i>Gadus morhua</i>) tropomyosin (C_TM) and mAb8F5 were used for assay development. The immunosignal was captured by two light sources, i.e., colorimetry (<i>cELISA</i>) and luminescence (<i>lELISA</i>). During assay validation, <i>cELISA</i> had a wider working range (1-250 ng/μL) than <i>lELISA</i> (0.5-62.5 ng/μL). However, <i>lELISA</i> showed better sensitivity because of its lower half-maximal inhibitory concentration (IC₅₀, 8.8 ng/μL) than <i>cELISA</i> (37.1 ng/μL). The optimal <i>cELISA</i> was applied to measure C_TM-equivalents in 21 bony fish samples (0.03-4.2 mg/g) and laboratory-spiked samples (recovery rates of C_TM-spiked shrimp samples ranged from 104.7 to 120.8%). <i>lELISA</i> was used to quantify 5.25 μg C_TM residues on four food contact surfaces (3.4-6.9 μg from C_TM residues and 3.2-4.8 μg from Atlantic cod were detected). This immunoassay has the potential to surveil undeclared allergenic bony fish residues and validate the hygiene of food contact surfaces.