McCall Jennifer R, Jacocks Henry M, Niven Susan C, Poli Mark A, Baden Daniel G, Bourdelais Andrea J
J AOAC Int. 2014 Mar-Apr;97(2):307-15. doi: 10.5740/jaoacint.sgemccall.
Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed a novel fluorescence-based binding assay for the brevetoxin receptor. Several fluorophores were conjugated to polyether brevetoxin-2 and used as the labeled ligand. Brevetoxin analogs were able to compete for binding with the fluorescent ligands. This assay was qualified against the standard radioligand receptor assay for the brevetoxin receptor. Furthermore, the fluorescence-based assay was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. The fluorescence-based assay was quicker, safer, and far less expensive. As such, this assay can be used to replace the current radioligand assay and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.
短裸甲藻毒素是一类在海洋双鞭毛藻短裸甲藻大量繁殖期间产生的梯架式聚醚毒素。食用接触过短裸甲藻大量繁殖的鱼类会导致神经性贝类中毒。短裸甲藻毒素的毒性作用是由于细胞膜中电压敏感钠通道（VSSCs）的激活。毒素的结合过去一直通过放射性配体竞争测定法来测量，该方法存在诸多困难。在本研究中，我们开发了一种用于短裸甲藻毒素受体的基于荧光的新型结合测定法。几种荧光团与聚醚短裸甲藻毒素 -2 偶联，并用作标记配体。短裸甲藻毒素类似物能够与荧光配体竞争结合。该测定法针对短裸甲藻毒素受体的标准放射性配体受体测定法进行了验证。此外，基于荧光的测定法用于确定短裸甲藻培养物粗提物中毒素的相对浓度，并确定雪卡毒素对 VSSCs 位点 5 的亲和力。基于荧光的测定法更快、更安全且成本低得多。因此，该测定法可用于替代当前的放射性配体测定法，并将成为未来研究各种配体与钠通道位点 5 结合亲和力实验的重要工具。
