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畜禽肉及饲料中氟苯尼考残留免疫分析方法研究
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作者单位
谢美婵,杨金易,李 然,肖治理,王 弘,徐振林,孙远明,沈玉栋 华南农业大学 食品学院/广东省食品质量安全重点实验室广东 广州 510642 
基金项目:十三五国家重点研发计划课题(2018YFC1602904); 国家自然科学基金资助项目(31871887); 广东省基础与应用基础研究项目(2019B1515210025, 2020A1515011238, 2018B030314005); 广东省企业科技特派员项目(GDKTP2020055300)
中文摘要:针对动物源性食品及饲料中氟苯尼考的残留问题,通过抗原制备、动物免疫和细胞融合筛选,成功得到可高特异性识别氟苯尼考的单克隆抗体,并建立了氟苯尼考的间接竞争酶联免疫分析(icELISA)方法。经单因素实验优化策略,确定最佳反应条件为:包被抗原质量浓度0.05 μg/mL,抗体质量浓度为0.1 μg/mL,最佳药物、抗体和二抗稀释液均为磷酸缓冲液(PBST),抗体稀释液吐温-20含量0.05%,竞争反应时间30 min,二抗质量浓度为0.167 μg/mL,二抗反应时间30 min。在该条件下,建立了氟苯尼考的icELISA 检测方法,其IC50为9.48 ng/mL,线性检测范围为1.75 ~ 51.36 ng/mL,检出限(LOD)达0.64 ng/mL,且与氯霉素等结构及功能类似物均无明显交叉反应,特异性良好。实际样品的加标回收率为88.1% ~ 107%,相对标准偏差(RSD)< 15%。将所建立的方法用于畜禽肉及饲料样品的检测,结果与HPLC-MS/MS判定结果一致,说明该方法适用于畜禽产品及饲料中氟苯尼考的残留检测。
中文关键词:氟苯尼考  畜禽肉及饲料  单克隆抗体  免疫分析
 
Study on a Monoclonal Antibody-based Immunoassay Method for Florfenicol in Animal Meat and Feed
Abstract:To monitor florfenicol residues in animal-derived foods and feeds, a monoclonal antibody with excellent specificity and sensitivity was prepared by using the antigen reported previously to immunize BALB/c mice and hybridoma technique. An indirect competitive enzyme-linked immunosorbent assay was established based on this antibody. The optimal conditions were finalized as follows: a coating antigen concentration of 0.05 μg/mL, an antibody concentration of 0.1 μg/mL, a competition reaction time of 30 min, a PBST diluent for analyte, primary antibody and secondary antibody with a Tween-20 of 0.05%, a secondary antibody concentration of 0.167 μg/mL and its reaction time of 30 min. Under the optimal conditions, the icELISA method for detection of florfenicol was established with an IC50 of 9.48 ng/mL, a linear range of 1.75-51.36 ng/mL and a limit of detection(LOD)of 0.64 ng/mL. There was no obvious cross-reactivity between the icELISA and its structural and functional analogs such as chloramphenicol, thiamphenicol, etc. The spiked recoveries for florfenicol in samples ranged from 88.1% to 107% with relative standard deviations(RSDs)less than 15%. The developed icELISA method was used to detect fifteen blind samples, of which four blind samples were found to contain florfenicol in different concentrations levels. These results were in good agreement with those of HPLC-MS/MS. Therefore, the developed icELISA exhibited a potential practicality in detection of florfenicol in animal meat and feed samples.
Key Words:florfenicol  animal meat and feed  monoclonal antibody  immunoassay
引用本文:谢美婵,杨金易,李 然,肖治理,王 弘,徐振林,孙远明,沈玉栋.畜禽肉及饲料中氟苯尼考残留免疫分析方法研究[J].分析测试学报,2021,40(9):1380-1385.
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