沙丁胺醇直接竞争ELISA法快速测定

Direct Competitive Enzyme-linked Immunosorbent Assay for the Rapid Determination of Salbutamol

建立沙丁胺醇的直接竞争ELISA(dcELISA)快速检测方法,采用高碘酸钠法制备辣根过氧化物酶标记沙丁胺醇单克隆抗体,棋盘滴定法确定包被抗原质量浓度和抗体稀释倍数,通过单因素试验,考察反应体系中表面活性剂、离子浓度、甲醇体积分数、pH值因素对dcELISA性能的影响,确定最优检测条件,同时考察方法的特异性。结果表明:酶标抗体克分子比为2.1时偶合物的滴度最高,最佳反应条件为包被抗原质量浓度1μg/mL,酶标抗体稀释2560倍,0.01mol/L、pH7.4的磷酸盐抗体稀释液(PBS)中含体积分数0.05%的Tween-20、0.5mol/LNaCl溶液和体积分数5%的甲醇时dcELISA具有最高的灵敏度和最好的稳定性,所建立方法的IC50为10.3ng/mL,检测限为0.049ng/mL,线性范围0.3～76.30ng/mL,批内变异系数13.8%,批间变异系数为22.38%,与克伦特罗交叉反应率为321.18%,与溴布特罗交差反应率为29.09%,与其他结构类似物没有明显交叉反应。本研究所建立的dcELISA可用于沙丁胺醇和克伦特罗的多残留检测。

A direct competitive enzyme-linked immunosorbent assay （dcELISA） was developed for rapidly determining salbutamol. Horseradish peroxidase-labeled salbutamol monoclonal antibodies were synthesized by sodium periodate method. The optimal coating concentration and antibody dilution were determined by chequerboard titration. The effects of surfactants,ion concentration,methanol content and pH on the sensitivity of dcELISA were investigated by single-factor experiments. The results showed that the optimal molar ratio of enzyme-labeled antibody was 2.1. The optimized assay conditions for both the highest sensitivity and the best stability were as follows：coating antigen concentration,1μg/mL; and dilution fold of antibody-enzyme conjugate in 0.01 mol/L pH 7.4 PBS dilution containing 0.05 % Tween-20,0.5 mol/mL NaCl,and 5% methanol,2560. The developed method presented an IC50 of 10.3 ng/mL,a detection limit of 0.049 ng/mL,a linear range of 0.3 to 76.30 ng/mL,with an intra-batch CV of 13.8 % and an inter-batch CV of 22.38%,and 321.18% and 29.09% cross-reactivity rates with clenbuterol and brombuterol,respectively,without notable cross-reactivity with other structural analogues of salbutamol. This study provides valuable experimental data for developing a commercial immunoassay kit for the rapid determination of multiresidues of salbutamol and clenbuterol.