抗玉米赤霉烯酮单克隆抗体免疫亲和柱的研究制备

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0．5ml溴化氰活化的4FF葡聚糖,350ugZEN单克隆抗体,柱容量为0．40ug。小麦样品添加ZEN标品60—300ug／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31．33～377．84ug／kg;信噪比为3：1时,检测下限为10．00ug／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。

Preparation of immunoaffinity column of zearalenone

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 microg when using 0.5 ml of CNBr activated Sepharose 4FF and 350 microg 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 microg/kg - 300 microg/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 microg/kg - 377.84 microg/kg. Detection limit based on a signal-to-noise ratio 3:1 was 10. 00 microg/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.