食品中四环素类残留的酶联免疫检测试剂盒的研制

Development of A New ELISA Kit for Tetracycline Residue Detection in Foods

目的:基于定量检测动物源性食品中四环素类抗生素的残留量,开发研制四环素类抗生素竞争酶联免疫(ELISA)检测试剂盒。方法:采用杂交瘤技术,筛选并克隆稳定分泌抗四环素(CEL)的杂交瘤细胞株,利用免疫BALB/c小白鼠制备抗四环素单克隆抗体。结果:logit/log拟合标准曲线为y=-2.3x+2.79,相关系数r=0.9971,线性检测范围为0.05～4.05ng/mL,半数抑制剂量(IC50)为0.293ng/mL,灵敏度为0.05ng/mL,检测限为3～5ng/mL;组织、牛奶、蜂蜜的添加回收率分别为(90±10)%、(85±10)%、(90±10)%;平均批内和批间变异系数均小于15%;四环素与金霉素等同类抗生素的交叉反应率大于60%,与其他类抗生素无交叉反应;此试剂盒在4℃可保存180d以上。结论:建立的试剂盒是符合技术指标的要求,可用于动物源性食品中四环素类抗生素残留的定量检测。

A competitive ELISA kit for tetracycline（TC）residues detection was developed by employing TC monoclonal antibody prepared by hybridoma technology using anti-TC monoclonal antibodies secreted by immunized BALB/c mouse.Performance tests showed that the logit/log calibration curve of the kit was y=-2.3x＋2.79（correlation coefficient,r=0.9971）,with a linear range from 0.05 to 4.05 ng/mL,the half inhibitory concentration（IC50）was 0.293 ng/mL,the sensitivity was 0.05 ng/mL and the detection limit ranged from 3 to 5 ng/mL.The recovery for spiked TC in muscle,milk,and honey was（90±10）%,（85±10）% and（90±10）%,with coefficient of variations（CV%）less than 15%,respectively.Cross-reactivity between TC and CTC was more than 60%,and there was no cross-reactivity between TC and other antibiotics.This ELISA Kit could be stored at 4℃for more than six months.