Sensitive and reliable micro-plate chemiluminescence enzyme immunoassay for okadaic acid in shellfish

Abstract

In this study, a highly sensitive and reliable analytical micro-plate chemiluminescence enzyme immunoassay (CLEIA) based on a monoclonal antibody (McAb) against okadaic acid (OA) was developed and validated for the detection of okadaic acid from a shellfish matrix. A competitive immunocomplex was formed through the binding of an immobilised antigen, OA, in analyzed samples and the McAb against OA. The conjugate OA–BSA was immobilised physically on a polystyrene micro-plate well as a solid phase antigen. Subsequently, free toxins in the analyzed samples competed with the solid phase antigen to bind the McAb against OA. The assay conditions, including the composition and pH of the coating solution, the dilution ratios and amounts of the McAb and the HRP-labelled goat anti-mouse IgG antibody, the time of the antibody-coating, incubation and chemiluminescence reactions and other relevant variables were studied and optimised. The optimised system allowed OA determination in a linear working range from 0.0098–10 μg kg⁻¹ (R = 0.99), and the calibration curve obtained for OA revealed a detection limit of 0.0098 μg kg⁻¹. Importantly, the CLEIA was approximately 10 times more sensitive than an ELISA using the same antibody. In addition, the intra- and inter-assay RSDs were both less than 10.0%. Moreover, this method was successfully applied to the evaluation of OA in seashells, with recoveries of 97.2%, 111.2% and 104.7%, respectively, for low-, medium- and high-concentration samples. Good recoveries were obtained from spiked food samples, and the results correlated well with those obtained using conventional indirect competition ELISA, indicating the potential utilisation of the CLEIA as a preliminary screening tool for analyzing OA contamination in shellfish.