Improvement of the efficiency and simplification of ELISA tests for rapid and ultrasensitive detection of okadaic acid in shellfish

Abstract

Okadaic acid (OA) is a marine toxin ingested by shellfish. The consumption of contaminated seafood causes diarrheic shellfish poisoning, responsible for gastrointestinal troubles in humans. The development of fast, reliable and sensitive methods for OA analysis is an evident necessity in order to guarantee the seafood safety and to protect human health.In this work, competitive indirect immunoassays were developed for the toxin detection. Indirect and direct labeling were used and compared. With indirect labeling, the unlabeled anti-OA antibody binds to the toxin and a labeled secondary antibody is used for the signal generation. Alternatively, using the direct labeling, the label is attached via a covalent bond to the anti-OA antibody. In this work, peroxidase was conjugated to the anti-OA antibody through a periodate activation. The peroxidase–antibody conjugate is used for both toxin recognition and signal generation. In the last case, ELISA test is simplified and the colorimetric competitive indirect immunoassay based on direct labeling is more sensitive to OA than the test based on indirect labeling. In optimized conditions, the IC50 value and detection limit were respectively 0.066 μg L−1 and 0.0061 μg L−1. Then, the colorimetric immunoassay based on direct labeling was validated with certified reference mussel samples, demonstrating the efficiency of the approach.