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 (Gadus morhua) tropomyosin (CTM) and mAb8F5 were used for assay development. The immunosignal was captured by two light sources, i.e., colorimetry (cELISA) and luminescence (lELISA). During assay validation, cELISA had a wider working range (1–250 ng/μL) than lELISA (0.5–62.5 ng/μL). However, lELISA showed better sensitivity because of its lower half-maximal inhibitory concentration (IC50, 8.8 ng/μL) than cELISA (37.1 ng/μL). The optimal cELISA was applied to measure CTM-equivalents in 21 bony fish samples (0.03–4.2 mg/g) and laboratory-spiked samples (recovery rates of CTM-spiked shrimp samples ranged from 104.7 to 120.8%). lELISA was used to quantify 5.25 μg CTM residues on four food contact surfaces (3.4–6.9 μg from CTM 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.