Development and Validation of a Dual-Mode (Colloidal Gold Screening + FRET Quantitative) Immunochromatographic Assay for Okadaic Acid in Shellfish Using a DTX1/2-Cross-Reactive Monoclonal Antibody
Description
Diarrhetic shellfish poisoning (DSP), primarily caused by okadaic acid (OA) and its analogues DTX1 and DTX2, remains a recurrent food safety concern in coastal monitoring worldwide. Current regulatory methods such as the mouse bioassay and HPLC-MS suffer from ethical concerns, low throughput, or laboratory dependence, whereas existing OA immunostrips remain largely qualitative, with limited sensitivity and no reported cross-reactivity against DTX congeners. Here, we report a dual-mode immunochromatographic platform based on a newly generated monoclonal antibody, mAb 4C9 (IgG1, IC50 2.59 ng/mL, cross-reactivity 45.4% with DTX1 and 47.8% with DTX2). The colloidal gold mode provides rapid visual screening with a cut-off of 15 ng/mL within 6 min, suitable for on-site qualitative judgment. The fluorescence-quenching (FRET) mode, pairing fluorescent microspheres (donor, Ex 468/Em 508 nm) with colloidal gold nanoparticles (acceptor) on a lab-built portable reader, achieves a limit of detection of 1.56 ng/mL, a linear range of 3.13–50 ng/mL (R² = 0.9921), and a 9-min runtime. In shellfish spike tests using OA-negative mussels at seven spike levels (0-50 ng/g tissue), recoveries ranged from 89.0% to 92.4% with relative standard deviations of 2.3-4.0%, and the measured values correlated tightly with theoretical concentrations (r = 0.9995). Both modes satisfy the EU regulatory threshold (16 µg/100 g) and the Japanese standard (20 µg/100 g) for DSP toxins. The dual-mode design supports a practical “screen-first, quantify-second” workflow for on-site DSP monitoring, and the intrinsic DTX1/2 cross-reactivity of mAb 4C9 enables semi-quantitative estimation of total DSP equivalents under a single OA calibration.
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Institutions
- Jinan UniversityGuangdong, Guangzhou