Sato Shigeru, Takata Yoshinobu, Kondo Sunaho, Kotoda Akiko, Hongo Naoto, Kodama Masaaki
J AOAC Int. 2014 Mar-Apr;97(2):339-44. doi: 10.5740/jaoacint.sgesato.
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.
开发了一种新的酶联免疫吸附测定（ELISA）试剂盒，用于定量分析贝类粗提物中麻痹性贝类毒素（PSP）的含量。基于诸如膝沟藻毒素2和3（GTX2,3）等C11 - O - 硫酸酯毒素与各种硫醇化合物之间的独特反应，制备了用于ELISA的抗原偶联物和新型抗体，随后与匙孔血蓝蛋白偶联。用于竞争ELISA的必要化合物、标记毒素以及在分析中替代石房蛤毒素的人工标准毒素，也通过相同技术制备。所得的ELISA能够识别所有测试的毒素成分；然而，诸如B和C毒素等氨基甲酰 - N - 硫酸盐衍生物以及诸如新石房蛤毒素和GTX1,4等N1 - OH毒素对该抗体的亲和力较低。在毒素成分之间观察到的抗体反应性差异妨碍了对贝类提取物中毒素含量的准确定量。为了解决这个问题，根据传统方法，通过温和的盐酸处理将前一种毒素成分转化为相应的氨基甲酸酯毒素。使用血红素作为催化剂，通过我们的原始方法将后一种毒素的N1 - OH还原为N1 - H。我们在此报告结合了预处理过程的新型ELISA试剂盒，该预处理过程可将毒素成分转化为有利于PSP毒素定量分析的形式。
