| The development and optimization of ELISA for the determination of tetrodotoxin |
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| 引用本文： | 周玉 李岩松 潘风光 柳增善 王哲. The development and optimization of ELISA for the determination of tetrodotoxin[J]. 中国人民解放军军医大学学报, 2007, 22(6): 347-351. DOI: 10.1016/S1000-1948(08)60016-7 |
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| 作者姓名： | 周玉 李岩松 潘风光 柳增善 王哲 |
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| 作者单位： | [1]Key Laboratory of Zoonoses, Ministry of Education, Institute of Zoonoses, Jilin University, Changchun130062, China [2]College of Light Industry and Economics ＆ Management, Jilin University, Changchun 130062, China [3]Institute of Farming and Veterinary, Jilin University, Changchun 130062, China |
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| 基金项目： | 吉林大学校科研和教改项目 |
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| 摘 要： | Objective： To optimize the ELISA for the determination of tetrodotoxin. Methods： A competitive enzyme-linked immunosorbent assay （ELISA） was used. In the ELISA, 100 μl antigen （1. 0 μg/ml） was coated on the microtiter plate for 60 min at 37 C or over night at 4 C. The plate was then washed 3 times with PBS-T for 3-5 s each time. The optimal incubation time for monoclonal antibody （mAb）, goat anti-mice IgG peroxidase conjugate and OPD were 30 min, 20 min and 10 min at 37 C, re- spectively. Results.. The detection limit is 0. 05 ng in each well. The curve was linear for TTX doses be- tween 5-5 000 ng/ml （0. 25-250 ng for every assay）. The linear regress equation was Y = 0. 30 88X-0.17 41 （R=0.99 01）. The average callback for TTX of muscles and gonads were 99.74% and 100.30%, respectively. The sensitivity of optimization ELISA was 5 times than traditional method and the time of 1.8 h were saved. Conclusion： The optimized ELISA is an ideal method for the determination of tetrodotoxin.
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| 关 键 词： | 河豚毒素 单克隆抗体 免疫检测法 最优化 |
| 收稿时间： | 2007-09-20 |
| 修稿时间： | 2007-11-20 |
The development and optimization of ELISA for the determination of tetrodotoxin |
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| ZHOU Yu,LI Yan-song,PAN Feng-guang,LIU Zeng-shan,WANG Zhe. The development and optimization of ELISA for the determination of tetrodotoxin[J]. Journal of Medical Colleges of PLA(China), 2007, 22(6): 347-351. DOI: 10.1016/S1000-1948(08)60016-7 |
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| Authors: | ZHOU Yu LI Yan-song PAN Feng-guang LIU Zeng-shan WANG Zhe |
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| Affiliation: | 1. Key Laboratory of Zoonoses, Ministry of Education, Institute of Zoonoses, Jilin University, Changchun 130062, China;2. College of Light Industry and Economics & Management, Jilin University, Changchun 130062, China;3. Institute of Farming and Veterinary, Jilin University, Changchun 130062, China;1. Centro de Investigacións Mariñas, Consellería do Medio Rural e do Mar, Xunta de Galicia, Aptdo. 13, 36620 Vilanova de Arousa, Spain;2. Department Biochemistry & Molecular Biology, University of Córdoba, Córdoba, Spain;3. Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC)/Hospital Universitario Reina Sofía/Universidad de Córdoba, Spain;1. Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong;2. State Key Laboratory of Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong;1. Biology Department & CESAM, University of Aveiro, Campus de Santiago, 3810-193, Aveiro, Portugal;2. ECIMAT-Universidade de Vigo, Illa de Toralla s/n, 36331 Coruxo-Vigo, Galicia, Spain;3. Department of Analytical Chemistry, University of the Basque Country (UPV/EHU), P.O. Box 644, E-48080 Bilbao, Basque Country, Spain;1. Núcleo de Engenharia de Pesca, Universidade Federal de Sergipe, CEP 49100-000 Aracajú, SE, Brazil;2. Embrapa Tabuleiros Costeiros, Bairro Jardins, Caixa Postal 44, CEP 49025-040 Aracajú, SE, Brazil;3. Departamento de Biologia Molecular, Universidade Federal da Paraíba, CEP 58051-900 João Pessoa, PB, Brazil;4. Maryland Department of Natural Resources, Cooperative Oxford Laboratory, 904 S. Morris Street, Oxford, MD 21654, USA;5. Virginia Institute of Marine Science, College of William & Mary, P.O. Box 1346, Gloucester Point, VA 23062, USA |
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| Abstract: | ObjectiveTo optimize the ELISA for the determination of tetrodotoxin.MethodsA competitive enzyme-linked immunosorbent assay (ELISA) was used. In the ELISA, 100 μl antigen (1.0 μg/ml) was coated on the microtiter plate for 60 min at 37 C or over night at 4 C. The plate was then washed 3 times with PBS-T for 3-5 s each time. The optimal incubation time for monoclonal antibody (mAb), goat anti-mice IgG peroxidase conjugate and OPD were 30 min. 20 min and 10 min at 37 C, respectively.ResultsThe detection limit is 0.05 ng in each well. The curve was linear for TTX doses between 5-5 000 ng/ml (0.25-250 ng for every assay). The linear regress equation was Y = 0.30 88X — 0.17 41 (R. = 0.99 01). The average callback for TTX of muscles and gonads were 99.74% and 100.30%, respectively. The sensitivity of optimization ELISA was 5 times than traditional method and the time of 1.8 h were saved.ConclusionThe optimized ELISA is an idealmethod for the determination of tetrodotoxin. |
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| Keywords: | tetrodotoxin monoclonal antibody ELISA |
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