Preparation of Polyclonal Antibody Against Domoic Acid and Establishment of Indirect Competitive ELISA Assay

Domoic acid (DA), the main component of amnesic shellfish poisoning (ASP), can be bio-concentrated in fish and shellfish and have toxic effects on humans through the transmission of food chains. DA, a small molecule hapten, was coupled to the carrier protein keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) with the activation ester method to prepare complete immunogen and coating antigen. DA-KLH and DA-BSA were successfully conjugated using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) and their conjugating ratios were 44 : 1 and 16 : 1, respectively, according to analyses with UV-visible spectrophotometry and SDS polypropylene gel electrophoresis (SDS-PAGE). The DA-specific titer of antiserum of 210 000 units/mL was obtained after immunizing the mice four times with DA-KLH. The optimal working concentrations and conditions were determined for detecting DA with the indirect competitive ELISA (ic-ELISA), and the DA detection standard curve was successfully plotted in the concentration range of 100 ng/mL~10 μg/mL. The rapid quantitative detection of DA with the established indirect competitive ELISA method might provide guidance for the development of the first-line seafood algae toxin detection kit and the red tide toxin monitoring system.