抗玉米赤霉烯酮单克隆抗体免疫亲和柱的研究制备

Preparation of immunoaffinity columm of zearalenone

目的研究制备抗玉米赤霉烯酮(Zearalenone,ZEN)单克隆抗体免疫亲和柱(IAC)。方法用辛酸硫酸铵法纯化抗 ZEN 单克隆抗体,将纯化好的单抗与溴化氰活化的4FF 葡聚糖偶联制备抗 ZEN 单克隆抗体免疫亲合柱。采用紫外扫描鉴定偶联反应,间接竞争酶联免疫吸附法及高效液相色谱法(HPLC)对制备好的免疫亲合柱进行评价。结果每根抗 ZEN 单克隆抗体免疫亲和柱,使用0.5 ml 溴化氰活化的4FF 葡聚糖,350 μg ZEN 单克隆抗体,柱容量为0.40 μg。小麦样品添加 ZEN标品60～300 μg/kg,回收率76.33%～90.10%,相对标准偏差6.68%～10.93%。使用本实验室制备的抗 ZEN 单克隆抗体免疫亲合柱建立了 IAC-HPLC 方法,并检测小麦、玉米、饲料样品30份,结果有17份样品为阳性,阳性率56.67%,样品中 ZEN 含量为31.33～377.84 μg/kg;信噪比为3:1时,检测下限为10.00 μg/kg。结论抗 ZEN 单克隆抗体免疫亲和柱能快速特异地将 ZEN 从样品中分离出来,并同时完成净化和浓缩步骤,是一种简便的净化方法。

Objective To prepare immunoaffinity column of zearalenone. Methods The zearalenone immunoaffinity column（ IAC ）was prepared by coupling CNBr-activated Sepharose 4 Fast Flow （4FF） with the anti-zearalenone monoclonal antibody which was purified by caprylic acid -ammonium sulfate method. The coupling reaction was identified by UV-absorbance measurements, and the IAC prepared was evaluated by indirect-competition ELISA and HPLC. Results The column capacity was determined to be 0. 40 ug when using 0. 5 ml of CNBr activated Sepharose 4FF and 350 ug of purified anti-zearalenone monoclonal antibody. The mean true recoveries were in the range 76. 33 % - 90. 10 % and RSD was 6. 68 % - 10. 93 % at levels of 60 ug/kg -300 ug/kg. 30 samples of wheat and maize were detected by the anti-ZEN IAC produced by the laboratory, 17 samples were observed to be contaminated in a comparable range from 31.33 ug/kg -377. 84 ug/kg. Detection limit based on a signal-to-noise ratio 3：1 was 10. 00 ug/kg for ZEN in wheat and maize. Conclusion IAC, a simple separating method which is used in ZEN extraction from cereals, is able to purify and condense ZEN in one step. The cost of detection can be lowered down because the IAC developed is hopefully to substitute the imported IAC.