沙拉沙星间接竞争化学发光酶联免疫检测方法的建立

Establishment of an Indirect Competitive Chemiluminescence Enzyme-linked Immunoassay for Sarafloxacin

为建立动物源性食品中沙拉沙星(SAR)残留的快速检测方法,本试验通过对盐酸沙拉沙星进行分子改造和偶联蛋白合成完全免疫原,免疫小鼠制备SAR单克隆抗体。通过棋盘法确定最佳包被原浓度及一抗稀释度,优化确定最佳反应条件,从而建立间接竞争化学发光酶联免疫(ic-CLEIA)检测方法,并通过灵敏度、精密度、交叉反应率、添加回收试验对该方法进行评价。紫外扫描结果显示,完全免疫原偶联成功;制备的SAR单克隆抗体效价达1∶4×106;包被原浓度为0.25μg/mL,抗体稀释比为1∶750000,4℃包被过夜,5%脱脂乳封闭,竞争孵育1 h,酶标二抗稀释比为1∶10000,孵育1 h为ic-CLEIA最佳反应条件;建立的ic-CLEIA方法标准曲线呈线性,线性范围为0.0625~10 ng/mL,R2=0.995,灵敏度IC50为1.45 ng/mL;其平均批内和批间变异系数均<10%;除与二氟沙星的交叉反应率达到98.08%以外,与其他氟喹诺酮类药物的交叉反应率均<8%,与其他非氟喹诺酮类药物均无交叉反应;SAR标准溶液的最低检测限(LOD)为0.32 ng/mL,SAR在鸡肉样品中的LOD为0.46μg/kg;添加回收率在88.3%~106.7%范围内,其变异系数≤12.2%。结果表明,本研究建立的ic-CLEIA方法检测速度快、灵敏度高,适用于动物源性食品中SAR残留的大量检测,为SAR残留检测提供了新的方法。

To establish a rapid detection method for sarafloxacin(SAR)drug residues in animal-derived foods,SAR monoclonal antibody was prepared by immunizing mice with sarafloxacin hydrochloride molecular modification and coupling protein synthesis complete immunogen.The checkerboard method was used to determine the optimal coating concentration(SAR-1-OVA)and primary antibody dilution,and optimize the optimal reaction conditions to establish an indirect chemiluminescence enzyme-linked immunoassay(ic-CLEIA).The method was evaluated by sensitivity,precision,cross-reaction rate,and additive recovery test.The results showed that the complete immunogen coupling was successful after UV scanning,and the prepared SAR monoclonal antibody titer reached 1∶4×106.The optimal reaction conditions of ic-CLEIA were as follows:Coating concentration was 0.25μg/mL,dilution of antibody was 1∶750000,coating at 4℃overnight,5%skim milk sealed,competitive incubation for 1 h,dilution of enzyme labeled secondary antibody was 1∶10000,incubation for 1 h.The standard curve was linear,the linear range was 0.0625 to 10 ng/mL,R2=0.995,the sensitivity IC50 was 1.45 ng/mL.The average intra-and inter-assay coefficients of variation were both<10%.Except the cross reaction rate with difloxacin was 98.08%,the cross reaction rates with other fluoroquinolones were<8%,and there was no cross reaction with other non fluoroquinolones.The minimum detection limit(LOD)of standard SAR was 0.32 ng/mL,and the LOD of SAR in chicken samples was 0.46μg/kg.The recovery rate of standard addition was in the range of 88.3%to 106.7%,and the coefficient of variation was≤12.2%.The above results showed that the method was fast and sensitive,was suitable for the large-scale detection of sarafloxacin residues in animal-derived foods,and this results provided a new method for the detection of sarafloxacin residues.