黃麴毒素B1單株抗體之製備並將其應用於酵素連結免疫吸附分析法及奈米金粒子免疫層析試紙分析法之開發

黴菌毒素是由黴菌所產生的次級代謝物，當人或動物誤食到被黴菌毒素污染的食物或飼料時，健康容易因此受到危害。黃麴毒素 (Aflatoxins) 是最常見的污染穀物作物之黴菌毒素，包含黃麴毒素B1、B2、G1、G2，在先前的研究中指出，黃麴毒素B1 (Aflatoxin B1；AFB1) 為最具毒性的黃麴毒素，其肝毒性會誘發急性肝炎或肝癌等病變，因此國際癌症研究中心 (International Agency for Research on Cancer；IARC) 將AFB1 歸類為 group 1，為人類絕對致癌物；美國食品藥物管理局限制其含量在食物及飼料中必須在 20 ppb 以下，而台灣訂為 15 ppb以下，歐盟則是 2 ppb。為了能夠快速準確的篩檢出食品及飼料中是否有被 AFB1 汙染，在此我們製備了 AFB1 的單株抗體，並利用抗體開發直接競爭型酵素連結免疫吸附分析法 (Competitive direct enzyme-linked immunosorbent assay；cdELISA) 以及奈米金粒子快速免疫層析試紙分析法。在 cdELISA 中，單株抗體 3F6G11的IC50 (抑制 50% AFB1-CMO-HRP 與抗體結合所需 AFB1 濃度) 為 0.0515 ng/mL，9C7C11H10的 IC50 為 0.0451 ng/mL；免疫層析試紙分析法對 AFB1的偵測極限則分別為 2 ng/mL (3F6G11)、1 ng/mL (9C7C11H10)，而利用單株抗體 3F6G11所開發出的 cdELISA 及 strip在樣品分析的結果上較有一致性。根據以上結果，我們成功利用單株抗體 3F6G11 建立了 AFB1 的 cdELISA 及 strip 這兩種快速免疫檢測方式，能夠簡單且快速的檢測食物及飼料中AFB1 的污染情形。

關鍵字

黴菌毒素；單株抗體；黃麴毒素 B1 ；酵素連結免疫吸附分析法；奈米金粒子免疫層析試紙；融合瘤

並列摘要

Mycotoxins are secondary metabolites produced by fungi. Foods or feeds which are contaminated by mycotoxins will harm the health of people or animals by ingestion. Aflatoxins, including AFB1、B2、G1、G2, were the most commonly occurred mycotoxins in cereal grain. According to previous research, AFB1 is the most toxic one which cause acute hepatitis or hepatic cancer by its hepatotoxicity. International Agency for Research on Cancer (IARC) has classified AFB1 as group 1, as a human carcinogen. Therefore, the U.S. Food and Drug Administration (FDA) enacted a regulatory limit of total aflatoxins in foods and feeds as 20 ppb. The maximum levels of total aflatoxins in Taiwan is 15 ppb. Moreover, AFB1 should below 2 ppb in the European Union. For correct and rapid detecting AFB1 in food and feeds, we produced AFB1-specific monoclonal antibody and applied it on a competitive direct enzyme-linked immunosorbent assay (cdELISA) and a gold nanoparticle based rapid immunochromatographic strip. The concentration causing 50% inhibition of binding AFB1-CMO-HRP to AFB1 antibodies (IC50) in cdELISA were found to be 0.0515 ng/mL in 3F6G11 and 0.0451 ng/mL in 9C7C11H10. The detection limit of immunochromatographic strip for AFB1 were 2 ng/mL in 3F6G11 and 1 ng/mL in 9C7C11H10, respectively. Results of analyzing food and feeds based on 3F6G11 monoclonal antibody showed a good agreement in cdELISA and strip. These data suggested that we successfully developed two kinds of rapid immunoassay by using AFB1 monoclonal antibody 3F6G11, cdELISA and strip assay, which could simply and rapidly detect AFB1 contamination in foods and feeds.

並列關鍵字

Mycotoxin ； Aflatoxin B1 ； monoclonal antibody ； ELISA ； Immunochromatographic Strip ； hybridoma

參考文獻

陳銘在, 傅曉萍, 施如佳, & 徐錦豐 (2009). 市售辣椒粉、胡椒粉及咖哩粉中黃麴毒素含量調查. 藥物食品檢驗局調查研究年報, 27, 278-283.

陳銘在, 傅曉萍, & 徐錦豐 (2009). 市售開心果、杏仁及瓜子中黃麴毒素含量調查. 藥物食品檢驗局調查研究年報, 27, 271-277.

Arafa, A. S., Bloomer, R. J., Wilson, H. R., Simpson, C. F., & Harms, R. H. (1981). Susceptibility of various poultry species to dietary aflatoxin. Br Poult Sci, 22(5), 431-436.

Butler, J. E., Feldbush, T. L., McGivern, P. L., & Stewart, N. (1978). The enzyme-linked immunosorbent assay (ELISA): a measure of antibody concentration or affinity. Immunochemistry, 15(2), 131-136.

Chu, F. S., & Ueno, I. (1977). Production of antibody against aflatoxin B1. Appl Environ Microbiol, 33(5), 1125-1128.

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黃麴毒素B1單株抗體之製備並將其應用於酵素連結免疫吸附分析法及奈米金粒子免疫層析試紙分析法之開發

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