Development of simple analyzing method for paralytic shellfish toxins by ELISA

尽管麻痹性贝类毒素(PSP毒素)已经发展了许多成熟的分析方法，但包括日本在内的许多国家仍将小鼠生物测定法作为监测双壳类毒素的官方方法。小鼠生物测定法是一种简单可靠的方法，但该方法在动物实验中存在许多问题。目前，需要一种简单、快速、可替代小鼠生物测定的方法。EL ISA Far PSP毒素是第一批候选毒素之一。为了制造出酶联免疫吸附试验所需的针对毒素的特异性抗体，将毒素与蛋白质等大分子物质结合在一起的半抗原。最近我们发现，在硫醇还原转化为黑曲霉毒素(STX)的过程中，GTX2，3的11位碳原子与硫醇的硫原子发生共价键合，形成稳定的毒素与硫醇的结合物。毒素和硫醇的偶联物使我们能够制备a…毒素通过硫醇与蛋白质结合的更多原素。将STX与谷胱甘肽(GSH)与牛血清白蛋白(BSA)的结合物免疫兔，制备PSP特异性抗体。所获得的抗体与所测试的所有PSP毒素组分的亲和力几乎相同，包括不与任何已报道的抗体发生反应的C毒素。然而，抗体效价太低，不能形成EILSA，可能是因为与蛋白质结合的毒素水平较低。因此，用乙二硫醇(EDT)代替谷胱甘肽(GSH)制备11位带有SH基团的STX，然后与马来酰亚胺引入的BSA反应，得到BSA-EDT-STX。BSA-EDT-STX免疫兔获得的抗体与PSF毒素的各组分具有较高的亲和力。该抗体对毒素具有较高的效价，为建立检测PSP毒素的酶联免疫吸附试验提供了可能。通过对扇贝提取物毒性为2MU/ml的实验，我们的酶联免疫吸附试验测定值为1.68MU/ml，这表明我们的酶联免疫吸附试验适用于双壳类毒素的常规监测，但还需要进一步的分析才能证实。较少

Although many sophisticated analyzing methods for paralytic shellfish toxins (PSP toxins) have been developed, mouse bioassay is still applied as official method for monitoring of bivalve toxicity in various countries including Japan. Mouse bioassay is a simple and reliable method, but many problems about animal experiments have been pointed out for this method. Currently a simple and rapid method which could be replaceable with mouse bioassay is needed. ELISA far PSP toxins is one of the first candidates. In order to make specific antibody against toxins required for ELISA, hapten antigen in which toxins are bound with high molecular weight substances such as protein. Recently we have found that in a process of reductive transformation of gonyautoxin (GTX) 2,3 to saxitoxin (STX) by thiols, a stable conjugate of toxin and thiol is formed in which carbon atom at 11 position of GTX2,3 binds with sulfur atom of thiol covalently. The conjugate of toxin and thiol enables us to prepare the a … More ntigen in which toxin is bound with protein via thiol. In a trial to make a specific antibody against PSP, conjugate of STX and glutathione (GSH) bound with bovine serum albumin (BSA) was immunized to rabbits. The obtained antibody showed almost equal affinity to all the PSP toxin components tested including C toxins which could not react with any antibody reported. However, titer of antibody was too low to develop EILSA, probably because of low level of toxins bound with protein. Thus ethane dithiol (EDT) was used instead of GSH to prepare STX with SH group at 11 position, then it was reacted with maleimido-introduced BSA to obtain BSA -EDT -STX. The antibody obtained from immunization of BSA -EDT -STX to rabbits showed high affinity to various components of PSF toxins. The antibody also showed high titer to toxins which made it possible to develop ELISA for PSP toxins. In a trial to measure the scallop extract with toxicity of 2 MU/ml, our ELISA showed the value of 1.68 MU/ml, indicating that our ELISA is applicable for the analysis of PSP toxins in regular monitoring of bivalve toxicity, though further analysis is necessary for the confirmation. Less