[关键词]
[摘要]
为建立快速检测猪尿等样品中盐酸克伦特罗(CL)的胶体金免疫层析方法,用柠檬酸三钠还原氯金酸制备了胶体金,将其标记抗CL单克隆抗体,制备了金标抗体;以CL-人血清白蛋白(HSA)为包被抗原、羊抗鼠IgG为质控线二抗,制成胶体金试纸。优化了胶体金颗粒粒径、标记抗体用量和pH值等各项参数,最终确定胶体金粒径为15 nm、每毫升胶体金溶液中添加20 ?g抗体、胶体金溶液pH值为7.4、金标抗体稀释液为添加0.1 %牛血清白蛋白(BSA)的0.05 mol/L磷酸盐缓冲液(pH 7.4)、金标抗体喷涂量为50 ?L/cm2、CL-HSA和羊抗鼠IgG的包被浓度分别0.5 mg/mL和2 mg/mL。研制的CL胶体金试纸检测限为3 ng/mL,与莱克多巴胺、沙丁胺醇等六种?-兴奋剂类药物无交叉反应。对42份猪尿样品的检测结果与市售酶联免疫(ELISA)试剂盒的符合率为100 %。试纸无需仪器辅助,操作简便,可在5 min内完成,适用于对CL残留进行现场检测。
[Key word]
[Abstract]
To develop colloidal gold immunochromatographic technique for the rapid detection of Clenbuterol (CL), colloidal gold was prepared by reduction of HAuCl4 using trisodium citrate. Anti-CL monoclonal antibodies (MAb) were then labeled with colloidal gold to obtain colloidal gold labeled MAb. The CL-human serum albumin (HSA) and goat anti-mouse IgG were immobilized on the lateral flow membrane to construct the strip. To obtain the best performance of the strip, several conditions were optimized as follows: the diameter of the colloidal gold particle 15 nm, the amount of MAb for labeling 20 ?g/mL, the pH value 7.4, the conjugate treating buffer 0.05 mol/L phosphate buffer (pH 7.4) containing 0.1% bovine serum albumin (BSA) and the thickness of colloidal gold-labeled CL antibody in use 50 ?L/cm2. In addition, the antigen and goat anti-mouse IgG were coated at 0.5 mg/mL and 2 mg/mL respectively. Under the optimum conditions, the limit of detection (LOD) of the strip was 3 ng/mL for swine urine. Cross-reaction tests showed that negligible cross-reactivity of the strip with six ?-Agonist compounds including ractopamine, salbutamol, etc. was detected. Forty-two samples of swine urine were detected by the developed strip and the results were confirmed by enzyme-linked immunosorbent assay (ELISA) kit. The accordance rate of the two methods was 100 %. The determination of CL by the strip did not need any instruments, and the total time for the determination of one sample was only 5 minutes. This method was applicable for the rapid determination of CL in the field.
[中图分类号]
[基金项目]
广东省自然科学基金(S2012010010323);广东省食品安全应急检测技术研究中心(2010A032000001-4);广东省教育部产学研结合项目(2011A090200029,2010A090200084)