以酵素免疫分析法和高效液相層析儀分析食品中赭麴毒素A之含量

Analysis of OTA in food by ELISA and HPLC methods

杞純鳳(Tsuen-Feng Chi)

指導教授 ： 余豐益

中山醫學大學/醫學院/醫學研究所/碩士(2005年)

https://doi.org/10.6834/CSMU.2005.00044

摘要

本實驗利用OTA -γ- globulin和OTA - KLH兩種不同的OTA接合物為抗原來免疫兔子 ， 進而收集免疫血清以獲得抗OTA 的多株抗體 ， 接著利用此一抗體來建立非直接型競爭酵素連結免疫吸附分析法 ( ciELISA ) 和直接競爭型酵素連結免疫吸附分析法 ( cdELISA ) ， 並用cdELISA的方法加以偵測咖啡及玉米類、麥類、豆類樣品中赭麴毒素A ( OTA ) 的含量 。 我們發現用OTA -γ- globulin 免疫後得到的抗體效價比以OTA - KLH免疫後得到的抗體效價來的高 。 因此，我們會以OTA -γ- globulin免疫兔子所得到的抗體為主發展出非直接和直接競爭酵素連結免疫吸附分析法的系統 。 在非直接競爭酵素連結免疫吸附分析系統中 ( ciELISA ) ， 此抗體對OTA 和赭麴毒素B ( OTB ) 以及赭麴毒素C ( OTC ) 的專一性反應以50 % 抑制濃度 ( IC50 ) 來表示 ， 其值分別為13 、 500 和2.7 ng/ml ； 此外在直接競爭酵素連結免疫吸附分析法中 ( cdELISA ) ， 此抗體對OTA 和 OTB 以及 OTC 的專一性反應以50 % 抑制濃度 ( IC50 ) 來表示 ， 其值分別為0.8 、100 和0.4 ng/ml 。 分析OTA的回收試驗 ( 10 ~ 250 ng/g ) ， 將毒素加入黃豆樣品中然後再用50 % 甲醇溶液萃取之 ， 再以cdELISA方法偵測OTA之平均回收率達85.9 % 。 我們以此免疫分析法比較不同萃取方法之萃取效果 ； 分析23種咖啡樣品 ( 以50 %甲醇溶液萃取 ) 中的OTA含量 ， 結果顯示23種樣品中有3個青豆樣品OTA含量低於20 ng/g ， 而有些咖啡樣品含量高於50 ng/g 。 以50 % 乙腈溶液所萃取出OTA的量比以50 %甲醇溶液萃出的還要高出約2 ~ 5倍 ; 玉米類、麥類、豆類樣品 ( 以70 % 甲醇溶液萃取 ) 中的OTA 含量 ， 結果顯示17個玉米類、麥類、豆類樣品中有11個樣品OTA含量介於16 ~ 160 ng/g 。 最後，我們同時輔以高效能液相層析儀 ( HPLC ) 分析咖啡、玉米類、麥類、豆類樣品中OTA之含量 ， 確認我們所建立的直接競爭型酵素免疫分析法 ( cdELISA ) 。 並且利用化學衍生法確定樣品中之OTA可被衍生成赭麴毒素A之甲基乙酯 ( OTA - methyl ester ) 。

關鍵字

赭麴毒素A ； 抗體 ； 酵素免疫分析法 ； 高效液相層析儀

並列摘要

Polyclonal antibodies for Ochratoxin A ( OTA ) were generated from rabbits after the animals had been immunized with either OTA - γ - globulin or OTA - keyhole limpet hemocyanin ( KLH ). A competitive indirect enzyme - linked immunosorbent assay ( ciELISA ) and a competitive direct ELISA ( cdELISA ) were used for the characterization of the antibodies and for analysis of OTA in coffee, corn, barley and bean samples. The antibody titers in the serum of rabbits immunized with OTA - γ - globulin were considerably higher than those in rabbits immunized with OTA - KLH. Therefore, the antibodies from the rabbits immunized with OTA - γ - globulin were further characterized. In the ciELISA, the concentrations causing 50 % inhibition ( IC50 ) of binding of OTA to the antibodies by OTA, Ochratoxin B ( OTB ) and Ochratoxin C ( OTC ) were found to be 13, 500, and 2.7 ng/ml, respectively ; In the cdELISA, the concentrations causing 50 % inhibition ( IC50 ) of binding of OTA - horseradish peroxidase to the antibodies by OTA, OTB, and OTC were found to be 0.8, 100, and 2.4 ng/ml, respectively. The recovery rate of OTA ( 10 ~ 250 ng/g ) added to soy bean samples and then extracted with 50 % aqueous methanol in the cdELISA was found to be 85.9 %. The coffee samples were subjected to two different solvent extraction and analyzed by cdELISA. The results of 50% aqueous methanol extracted showed that 3 of the 23 coffee samples examined were contaminated with OTA at levels lower than 20 ng/g ; Some of the others examined were contaminated with OTA at levels higher than 50 ng/g. The samples extracted with 50 % aqueous acetonitrile were 2 ~ 5 folds higher than samples extracted with 50 % aqueous methanol ; Analysis of OTA in corn, barley and bean samples with 70 % aqueous methanol showed that 11 of 17 corn, barley and bean samples examined were contaminated with OTA at levels between 16 ~ 160 ng/g. We also analyzed OTA in coffee, corn, barley, and bean sample with HPLC method to confirm the efficacy of cdELISA. The OTA in samples were further assured by chemical derivatization of OTA into its methyl ester.

並列關鍵字

Ochratoxin A ； Antibodies ； ELISA ； HPLC

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