Development of simple analyzing method for paralytic shellfish toxins by ELISA

2003 Fiscal Year Final Research Report Summary

• Project/Area Number: 12556035
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 展開研究
• Research Field: Fisheries chemistry
• Research Institution: Kitasato University
• Principal Investigator: SATO Shigeru Kitasato University, School of Fisheries Sciences, Associate Professor, 水産学部, 教授 (40050588)
• Co-Investigator(Kenkyū-buntansha): SHINAGAWA Kunihiro Iwate University, Faculty of Agriculture and Graduate School of Agriculture, Professor, 農学部, 教授 (60133906) ; SAKAI Ryuichi Kitasato University, School of Fisheries Sciences, Associate Professor, 水産学部, 助教授 (20265721) ; 佐藤 繁 北里大学, 水産学部, 助教授 (20170748)
• Project Period (FY): 2000 – 2003
• Keywords: paralytic shellfish toxins / ELISA / hapten / antigen / thiol / ethanedithiol / gonyautoxin / saxitoxin

Research Abstract

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 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.