Chemiluminescent Receptor Binding Assay for Ciguatoxins and Brevetoxins Using Acridinium Brevetoxin-B2

Abstract

Ciguatera is the term for poisoning resulting from eating fish from tropical or subtropical regions. The causative toxins collectively named ciguatoxins (CTXs) widely differ in structures depending on their geographic origins, which range from the Pacific Ocean and the Indian Ocean to the Caribbean Sea. Neurotoxic shellfish poisoning (NSP) is caused by the ingestion of bivalve shellfish contaminated with brevetoxins (BTXs). Structurally, both CTXs and BTXs consist of fused ether rings aligned in a ladder shape. Pharmacologically, they bind at the same site (site-5) of voltage-gated sodium channels. However, the great structural diversity and the rare availability of reference toxins hinder LC-MS and ELISA methods, which operate on structure-based recognition. In this study, we prepared a chemiluminescent ligand, acridinium BTXB2 (ABTX), and tested its suitability for use in competitive binding assays to detect CTXs and BTXs. The affinity of ABTX to the rat brain synaptosome estimated by Ki (1.66 pM) was approximately two-fold higher than that of PbTx-3 (BTX3). In addition, the equilibrium dissociation constant (KD) was 0.84 nM, the maximum number of binding was 6.76 pmol toxin/mg protein, and the detection limit was 1.4 amol. The assays performed on samples spiked with CTX3C or BTXB4 (N-palmitoylBTXB2) at 0.2−1.0 ng CTX/g fish flesh, and 200−800 ng BTXB4/g shellfish showed a linear relationship between the theoretical and observed toxin amounts.

Keywords

• ciguatera
• ciguatoxins
• neurotoxic shellfish poisoning
• brevetoxins
• acridinium ligand
• chemiluminescent binding assay
• synaptosome