Development of simple analyzing method for paralytic shellfish toxins by ELISA

尽管麻痹性贝类毒素（PSP毒素）的分析方法已经发展成熟，但包括日本在内的许多国家仍然采用小鼠生物测定法作为双壳贝类毒性监测的官方方法。小鼠生物测定法是一种简便、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。ELISA法是检测PSP毒素的首选方法之一。为了制备ELISA所需的针对毒素的特异性抗体，需要将毒素与蛋白质等高分子量物质结合的半抗原。最近我们发现，在用巯基还原gonyautoxin（GTX）2，3生成石房蛤毒素（STX）的过程中，GTX 2，3的11位碳原子与巯基的硫原子共价结合，形成了一种稳定的毒素-巯基结合物。毒素和硫醇的结合物使我们能够制备a 关于我们 其中毒素通过巯基与蛋白质结合的抗原。用STX与谷胱甘肽（GSH）结合物与牛血清白蛋白（BSA）免疫家兔，制备特异性抗PSP抗体。所获得的抗体对所有测试的PSP毒素组分显示几乎相等的亲和力，包括不能与任何报道的抗体反应的C毒素。然而，抗体的滴度太低，不能发展EILSA，可能是因为与蛋白结合的毒素水平低。以乙二胺二硫醇（EDT）代替谷胱甘肽（GSH）制备11位含巯基的STX，再与马来酰亚胺基引入的牛血清白蛋白（BSA）反应，得到BSA-EDT-STX。BSA-EDT-STX免疫家兔获得的抗体对PSF毒素的各种成分都有很高的亲和力.该抗体对毒素也显示出高滴度，这使得有可能开发PSP毒素的ELISA。在测定扇贝提取物的毒性为2 MU/ml的试验中，我们的ELISA显示的值为1.68 MU/ml，表明我们的ELISA适用于PSP毒素的分析，但需要进一步的分析来确认。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