β2-受体激动剂类药物多残留间接竞争ELISA检测方法的建立

Development of an indirect competitive ELISA for detection of multiple residues of β2-agonist drugs

【目的】建立β2-受体激动剂类药物多残留间接竞争ELISA检测方法,为动物食源性β2-受体激动剂类药物残留的快速检测提供技术支持。【方法】以沙丁胺醇多克隆抗体为基础,优化间接竞争ELISA的检测条件,建立一种快速检测猪尿中沙丁胺醇、卡布特罗、西布特罗、克伦特罗、溴布特罗、马布特罗、克伦潘特、可尔特罗、吡布特罗、马喷特罗、比托特罗与妥布特罗等12种β2-受体激动剂类药物的间接竞争ELISA检测方法。【结果】建立的β2-受体激动剂类药物间接竞争ELISA检测方法的最佳反应条件为:37℃,抗原以1∶10000倍包被1 h,抗体以1∶40000稀释作用40 min,酶标抗体以1∶20000稀释作用40 min。采用建立的间接竞争ELISA最佳反应条件对猪尿中的β2-受体激动剂类药物残留进行检测,结果表明12种β2-受体激动剂类药物在0.1~20μg/L范围内线性关系良好,相关系数均大于0.952;检出限为0.15μg/L,定量限为0.5μg/L;在0.5与1.0μg/L的添加浓度下,回收率为68.4%~114.2%,批内、批间RSD分别为0.4%~10.1%和0.4%~12.5%。【结论】所建立的β2-受体激动剂类药物残留ELISA检测方法操作简单、快速,灵敏度高,适用于猪尿中β2-受体激动剂药物的筛选与检测。

【Objective】An indirect competitive ELISA method was established for detection ofβ2-agonists multiple residues to provide support for the detection ofβ2-agonists in animal-origin food.【Method】Salbutamol polyclonal antibody was used and a rapid detection method was established for the test of salbutamol,capbuterol,cibuterol,clenbuterol,brombuterol and marbuterol,clenpentero,colterol,pirbuterol,mapenterol,bitolterol and tulobuterol in pig urine.【Result】The optimization conditions for the established indirect competitive ELISA method were temperature 37℃,antigen coated with 1∶10000 times for 1 h,the antibody diluted with 1∶40000 for 40 min,and the enzyme labeled antibody diluted with 1∶20000 for 40 min.The results of detectingβ2-agonist residues in pig urine showed that the 12 drugs had a good linear relationship within the range of 0.1―20μg/L,and the correlation coefficients were all greater than 0.952.The detection limit of pig urine sample was 0.15μg/L and the quantitative limit was 0.5μg/L.When 0.5 and 1.0μg/L of standard solutions were added to pig urine,the recovery rates were 68.4%-114.2%,the intra-RSDs were 0.4%―10.1%and the inter-RSDs were 0.4%―12.5%.【Conclusion】The established method is simple,rapid and sensitive,and it is suitable for the detection ofβ2-agonists in pig urine.