玉米赤霉烯酮单克隆抗体的制备及间接竞争ELISA检测方法的建立

Preparation of anti-zearalenone monoclonal antibodies and development of an indirect competitive ELISA for zearalenone

摘要:

玉米赤霉烯酮具有较强的生物毒性, 检测谷物中的玉米赤霉烯酮在食品和饲料安全中具有重要的作用。将玉米赤霉烯酮与牛血清白蛋白的偶联物免疫BALB/c小鼠制备单克隆抗体, 并建立基于单克隆抗体的酶联免疫法作为检测玉米赤霉烯酮的方法。结果共筛选到4株抗玉米赤霉烯酮单克隆抗体, 3株抗体亚类为IgG1, 1株为IgG2b。选择其中的一株杂交瘤细胞2C9制备小鼠腹水, 纯化后测定了抗体效价为1/40 000。以此单抗建立的间接竞争ELISA方法, 其半数抑制率(IC50)为1.90 ng/mL, 检测限(IC10)为0.051 ng/mL, 检测区间(IC20?IC80)为0.115?13.900 ng/mL; 且对玉米赤霉烯酮有很好的特异性。回收率检测在样品含1.46?93.80 μg/kg时回收率为96.5%?113.0%。本实验建立的检测方法可用于多种谷物及饲料样本中玉米赤霉烯酮的检测。

关键词:玉米赤霉烯酮, 单克隆抗体, 酶联免疫法, 回收率

Abstract:

Zearalenone (ZEN) is a high toxic mycotoxin. How to detect ZEN from food and feed is important and challenging. Monoclonal antibody was prepared by means of immunization in mice with conjugates of ZEN and bovine serum albumin (BSA), and enzyme-linked immunosorbent assay for ZEN based on monoclonal antibodies was developed. Four hybridoma cell strains secreting anti-zearalenone monoclonal antibodies were cloned. The subclasses of the monoclonal antibody were characterized as Ig G1 for three and Ig G2b for one. One of the cell strains 2C9 was used to prepare ascites for its high bioactivity. The titer of purified ascites was 1/40 000. The indirect-competitive ELISA was developed with good specificity for ZEN, the half maximal inhibitory concentration (IC50) is 1.90 ng/mL, the detection limit is 0.051 ng/mL, and the detection range is 0.115?13.9 ng/mL. The recovery rate could get to 96.5%?113% while the sample containing ZEN 1.46?93.8 μg/kg. The method for detection was de-veloped, which could be applied to detect ZEN from a variety of grain and feed samples.

Key words:Zearalenone, Monoclonal antibody, Enzyme-linked immunosorbent assay, Recovery rate