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Development of simple analyzing method for paralytic shellfish toxins by ELISA

麻痹性贝类毒素ELISA简易分析方法的建立

基本信息

批准号：
12556035
负责人：
SATO Shigeru
金额：
$ 7.55万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2003
项目状态：
已结题

项目摘要

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

虽然麻痹性贝类毒素（PSP毒素）的分析方法已经发展出许多成熟的方法，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贝类毒性的官方方法。小鼠生物测定法是一种简便、可靠的方法，但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法检测PSP毒素是首批候选物之一。为了制造ELISA所需的针对毒素的特异性抗体，毒素与高分子量物质（如蛋白质）结合的半抗原。最近我们发现，在硫醇还原转化gonyautoxin (GTX) 2,3为saxitoxin （STX）的过程中，gtx2,3的11位碳原子与硫醇的硫原子共价结合，形成了一种稳定的毒素与硫醇的共轭物。毒素与硫醇的结合使我们能够制备出毒素通过硫醇与蛋白质结合的抗原。将STX与谷胱甘肽（GSH）结合的牛血清白蛋白（BSA）偶联物免疫家兔，制备抗PSP特异性抗体。所得抗体与所有PSP毒素成分具有几乎相等的亲和力，其中包括C毒素，该毒素不能与任何报道的抗体发生反应。然而，抗体滴度太低，可能是由于与蛋白质结合的毒素水平较低，无法发生EILSA。因此，用乙烷二硫醇（EDT）代替谷胱甘肽制备SH基团位于11位的STX，然后与马来酰氨基引入的牛血清白蛋白反应得到BSA -EDT -STX。兔免疫BSA -EDT -STX获得的抗体对PSF毒素的多种成分具有较高的亲和力。该抗体对PSP毒素也有较高的效价，为建立PSP毒素的ELISA检测提供了可能。在毒性为2 MU/ml的扇贝提取物试验中，我们的ELISA检测结果为1.68 MU/ml，说明我们的ELISA可用于常规双壳类动物毒性监测中PSP毒素的分析，但还需进一步分析证实。少