開發酵素免疫分析法及奈米金粒子快速免疫試紙來檢測鐮孢毒素B1,黃麴毒素M1及微囊藻毒-LR

小分子毒素常見於日常生活中，誤食這些小分子毒素容易對我們的健康造成危害，本研究以伏馬鐮孢毒素B1 (fumonisin B1, FB1)、黃麴毒素M1 (aflatoxin M1, AFM1)與微囊藻毒LR (Microcystin-LR, MCY-LR)為研究主題，希望建立一套有效的檢測方式來檢測這三種小分子毒素。我們以抗體¬-抗原之間具有專一性的特性，利用這三種小分子毒素的抗體來建立其檢測方式。首先，我們將這三種小分子毒素分別接合至不同的載體蛋白質使其具有免疫性，並且以此接合物免疫兔子，分別成功的製備了針對這三種小分子毒素的多株抗體，並且開發出分別檢測FB1、AFM1與MCY-LR這三種小分子毒素的直接競爭型酵素免疫吸附分析法(cdELISA)，其抑制50 %抗原-酵素接合物與抗體結合所需的抗原濃度(IC50)分別為0.71、0.11與0.17 ng/mL。由於cdELISA需要在實驗室中才能運作，為了開發一套適合一般大眾的檢測方式，我們另外開發快速免疫層析試紙(immunostrip)用於檢測這三種小分子毒素，我們成功的開發出檢測FB1與AFM1的immunostrip，其偵測限制量(detection limit)皆為5 ng/mL。利用cdELISA與immunostrip檢測樣品時，兩種檢測方式的結果具有良好的一致性，我們開發的cdELISA及immunostrip能快速且有效的檢測樣品中的小分子毒素，以避免大眾受到這些小分子毒素的危害。

關鍵字

伏馬鐮孢毒素B1 ；黃麴毒素M1 ；微囊藻毒LR ；酵素免疫吸附分析法；免疫試紙；抗體；奈米金粒子

並列摘要

Mycotoxins and phycotoxins are low-molecular-weight nonimmunogenic toxins, which are frequently found in our environment. They are potential hazards to human and animal health. This study focused on three different kinds of toxins, fumonisin B1 (FB1), aflatoxin M1 (AFM1) and microcystin-LR (MCY-LR), and developed an one efficient method to detect the toxin. We designed the method based on the specificity of antibody and antigen. For producing the polyclonal antibodies against each FB1, AFM1 and MCY-LR, each toxin was conjugated to the different carrier proteins and immunized the rabbits, respectively. A direct competitive enzyme-linked immunosorbent assay (cdELISA) was used for the characterization of the antibodies and for analysis of the toxins in the samples. The concentrations causing 50 % inhibition of binding of toxin-horseradish peroxidase (HRP) to the antibodies by FB1, AFM1 and MCY-LR in the cdELISA were found to be 0.71, 0.11 and 0.17 ng/mL, respectively. Because cdELISA have to be analysed in the laboratory, it is needed to develop an assay that could be widely used in on-site screening of samples. Therefore, our study is focused on the development of gold-nanoparticle based rapid immunochromatographic strip, which is simpler and less time-consuming than cdELISA. In this study, both gold-nanoparticle based rapid immunochromatographic strips suitable for on-site FB1 and AFM1 determination were established with the detection limit of 5 ng/mL. Results of samples analysis obtained from gold-nanoparticle based rapid immunochromatographic strips were in a good agreement with those obtained from cdELISA. The cdELISA and gold-nanoparticle based rapid immunochromatographic strip can be rapid and efficient to detect the toxins in the samples.

並列關鍵字

Fumonisin B1 ； Aflatoxin M1 ； Microcustin-LR ； ELISA ； immunostrip ； antibody ； gold-nanoparticle

參考文獻

Azevedo, S. M., Carmichael, W. W., Jochimsen, E. M., Rinehart, K. L., Lau, S., Shaw, G. R., and Eaglesham, G. K. (2002). Human intoxication by microcystins during renal dialysis treatment in Caruaru-Brazil. Toxicology 181-182, 441-446.

Barco, M., Rivera, J., and Caixach, J. (2002). Analysis of cyanobacterial hepatotoxins in water samples by microbore reversed-phase liquid chromatography-electrospray ionisation mass spectrometry. J Chromatogr A 959, 103-111.

Carmichael, W. W. (1992). Cyanobacteria secondary metabolites--the cyanotoxins. J Appl Bacteriol 72, 445-459.

Carmichael, W. W. (1994). The toxins of cyanobacteria. Sci Am 270, 78-86.

Chen, C. Y., Li, W. J., and Peng, K. Y. (2005). Determination of aflatoxin M1 in milk and milk powder using high-flow solid-phase extraction and liquid chromatography-tandem mass spectrometry. J Agric Food Chem 53, 8474-8480.

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開發酵素免疫分析法及奈米金粒子快速免疫試紙來檢測鐮孢毒素B1,黃麴毒素M1及微囊藻毒-LR

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