孔雀石绿单克隆抗体的制备及直接竞争ELISA方法的建立.pdfVIP

※分析检测 食品科学 2009, Vol. 30, No. 24 283 孔雀石绿单克隆抗体的制备及直接竞争ELISA 方法的建立 1 2 3 1 , 李晓丽 ，陈雪岚 ，刘春梅 ，熊勇华 * ( 1.南昌大学食品科学与技术国家重点实验室，江西 南昌 330047 ；2 .江西师范大学生命科学学院，江西 南昌 330022 ；3.无锡中德伯尔生物技术有限公司，江苏无锡 2 14 174) 摘 要：通过合成羧基化孔雀石绿，采用活泼酯法制备孔雀石绿免疫抗原CMG-B SA 、检测抗原CMG-OVA 。采 用CMG-B SA 免疫 BAL B/c 小鼠，经融合、筛选和克隆化获得了一株稳定的抗孔雀石绿单克隆抗体，并建立了检 测孔雀石绿的直接竞争ELI SA 方法。ELI SA 方法的最佳操作参数：孔雀石绿抗体稀释倍数1:16000( 1mg/ml) ，酶标 抗原(CMG-HRP)使用质量浓度0 .2 μg/ml 。该方法具有良好的灵敏度和特异性，IC50 值0 .3ng/ml ，最低检测灵敏度 达到0.05ng/ml ，标准曲线方程为y ＝－20.888x ＋8 1.423(R2 ＝0.9813) 。 关键词：孔雀石绿；单克隆抗体；直接竞争E L I S A Preparation of Monoclonal Antibody of Malachite Green and Establishment of Direct Competitive ELISA 1 2 3 1, LI Xiao-li ，CHEN Xue-lan ，LIU Chun-mei ，XIONG Yong-hua * (1. State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China；2. College of Life Science, Jiangxi Normal University, Nanchang 330022, China ；3. Wuxi Zodolabs Biotech Co. Ltd., Wuxi 2 14 174, China) Abstract ：Carboxyl-malachite green (CMG) was synthesized and immunogen was prepared by coupling CMG with carrier protein BSA through an ester activation method. BALB/c mice were subj ected to inj ection of CMG-BSA and a hybridoma cell line secreting monoclonal antibody again st MG was con structed after fusion , screening and cloning experiments. A direct competitive ELISA for detection MG was established with 0.2 μg/ml CMG-HRP and 1:16000 of antibody dilution ratio. This method exhibited high sensitivity and excellent specificity for detecting MG. The standard regression equation of this