Quantitative ELISA Kit for Paralytic Shellfish Toxins Coupled with Sample Pretreatment
Quantitative ELISA Kit for Paralytic Shellfish Toxins Coupled with Sample Pretreatment
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DOI：
10.5740/jaoacint.sgesato
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发表时间：
2014-03-01
影响因子：
1.6
通讯作者：
Kodama, Masaaki
中科院分区：
文献类型：
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作者：
Sato, Shigeru;Takata, Yoshinobu;Kodama, Masaaki
研制了一种新的测定麻风性贝毒(PSP)粗提物中PSP毒素水平的酶联免疫吸附试验试剂盒。基于C11-0-硫酸盐毒素2和3(GTX2，3)与不同的硫醇类化合物发生独特的反应，然后偶联到锁孔帽状血蓝蛋白上，制备了一种用于制备抗原的偶联物和一种用于ELISA的新型抗体。竞争酶联免疫吸附试验所需的化合物、标记毒素和替代分析中的肉毒素的人工标准毒素也是通过相同的技术生产的。由此产生的酶联免疫吸附试验识别所有测试的毒素成分；然而，氨基甲酰-N-硫酸酯衍生物，如B和C毒素，以及N1-OH毒素，如新STX和GTX1，4对抗体的亲和力较低。在毒素成分之间观察到的抗体反应性的差异阻碍了对贝类提取物中毒素数量的准确量化。为了解决这个问题，按照传统的方法，通过温和的盐酸处理，将原来的毒素组分转化为相应的氨基甲酸酯毒素。后一种毒素的N1-OH还原为N1-H是在我们最初的方法中以氯化血红素为催化剂进行的。本文报道了新的酶联免疫吸附试剂盒与前处理过程相结合，以转化有利于PSP毒素定量分析的毒素组分。
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 C11-0-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 HCI 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.