Quantitative ELISA kit for paralytic shellfish toxins coupled with sample pretreatment

A new ELISA kit to quantitate the level of paralytic shellfish poisoning (PSP) toxins in crude shellfish extracts was developed. A conjugate for preparing antigen and a novel antibody used in the ELISA was prepared based on the unique reactions between C11-O-sulfate toxins such as gonyautoxins 2 and 3 (GTX2,3) and various thiol compounds, followed by coupling to keyhole limpet hemocyanin. The compounds necessary for competitive ELISA, labeled toxin and an artificial standard toxin to replace saxitoxin in the analysis, were also produced by the same techniques. The resulting ELISA recognized all the toxin components tested; however, carbamoyl-N-sulfate derivatives such as B and C toxins and N1-OH toxins such as neoSTX and GTX1,4 showed low affinity to the antibody. The difference in the reactivity of the antibody observed among the toxin components prevents accurate quantification of the toxin amounts in shellfish extracts. To address this problem, the former toxin components were transformed to corresponding carbamate toxins by mild HCl treatment according to a conventional method. The reduction of N1-OH of the latter toxins to N1-H was performed by our original method using hemin as a catalyst. We report here the new ELISA kit coupled with the pretreatment process to transform the toxin components favorable for the quantitative analysis of PSP toxins.