摘要
建立蜂蜜中氯霉素直接竞争酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)检测方法。以抗氯霉素单克隆抗体为包被原,氯霉素-辣根过氧化物偶联物为标记物,3,3’,5,5’-四甲基联苯胺作显色底物,对其主要影响因素,如标准品稀释液、包被温度、包被抗体稀释倍数等6个因素进行优化,同时还对该方法的准确性和稳定性进行考察。结果表明:所建立的直接竞争ELISA标准曲线方程式为y=-17.425x+97.509,相关系数R^2为0.991 2,IC_(50)值为0.63 ng/mL,检出限和线性检测范围分别为(0.04±0.01)ng/mL和0.10~4.17 ng/mL;另外整个检测反应过程耗时约为40 min。蜂蜜样品的检出限和定量限分别为0.15 ng/g和0.33 ng/g,添加回收率在97.58%~100.94%之间,批内变异系数和批间变异系数均小于11%,表明该方法具有高精确度和稳定性。
In this study,a rapid,accurate and stable direct competitive ELISA(dcELISA)for the detection of chloramphenicol residue in honey was developed.The anti-chloramphenicol monoclonal antibody was used as coating antigen,chloramphenicol-horseradish peroxidase conjugate as marker and 3,3’,5,5’-tetramethyl benzidine(TMB)as chromogenic substrate.Six main experimental conditions,including buffer for chloramphenicol standard,coating temperature,and dilution of coating antibody were optimized.In addition,the accuracy and stability of the method were also investigated.The obtained standard curve equation was y=-17.425x+97.509(R2=0.991 2),and the IC50 was 0.63 ng/mL.The limit of detection(LOD)and linear rang were(0.04±0.01)ng/mL and 0.10-4.17 ng/mL,respectively.The whole detection process required about 40 min.In honey samples,the LOD,LOQ and spiked recovery were 0.15 ng/g,0.33 ng/g and 97.58%-100.94%,respectively.The intra-batch and inter-batch coefficients of variation were both less than 11%.These results indicated that the developed dcELISA method possessed high accuracy and stability.
作者
刘姚
韦倩妮
王弘
杨金易
孙远明
徐振林
沈兴
李向梅
雷红涛
LIU Yao;WEI Qianni;WANG Hong;YANG Jinyi;SUN Yuanming;XU Zhenlin;SHEN Xing;LI Xiangmei;LEI Hongtao(Guangdong Provincial Key Laboratory of Food Quality and Safety,National-Local Joint Engineering Research Center for Processing and Safety Control of Livestock and Poultry Products,Guangzhou 510642,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2018年第16期336-342,共7页
Food Science
基金
“十三五”国家重点研发计划重点专项(2017YFC1601700)
广东省农业发展和农村工作专项(2017LM2152)
广州市科技计划项目(2016201604030004)。
关键词
氯霉素
蜂蜜
直接竞争ELISA
优化
chloramphenicol
honey
direct competitive ELISA
optimization
