AFP纳米金侧流免疫层析快速检测试纸的研制 方立超[ ]，程平，蒋丽莉 .DOC

AFP纳米金侧流免疫层析快速检测试纸的研制 方立超(，程平，蒋丽莉，黄辉，李艳，贺娟，邓均，郑峻松 （中国人民解放军第三军医大学医学检验系暨药学院临床检验学教研室 重庆，400038） [摘要] 目的 建立本教研室纳米金侧流免疫快速检测试纸研发的技术平台。方法 通过柠檬酸三钠还原法制备纳米金颗粒。以（A520－A580）为纵坐标，相应的金标记参数（pH、抗体量）为坐标曲线，曲线拐点为选择参数。标记缓冲体系的选择主要标记过程中确定根据免疫金的释放程度和速度选择合适的硝酸纤维素膜和作为结合垫的玻璃纤维素膜。组装检测临床血清标本，比较与ELISA检测的符合率。结果 AFP抗体I最适标记pH为8.5，最适抗体标记量为78(g/ml纳米金，标记缓冲体系为pH8.5 M Tris-HCl溶液硝酸纤维素膜为whatman Immunopore RP，以玻璃纤维素膜Ahlstrom8964作为结合垫和样品垫膜封闭洗涤后在26℃ 湿度26%条件下干燥1h，结合垫在26℃ 湿度26%条件下干燥2h。组装检测临床血清标本阳性检出率与ELISA法符合率为0%。 结论成功建立了本教研室纳米金侧流免疫快速检测试纸研发的技术平台，适用于病原微生物感染及肿瘤特异性标志物的现场快速检测。 Department of Clinical Laborarory Medicine，College of Pharmacy and Laboratory Medicine，Third Military Medical University, Chongqing 400038, China) [Abstract] Objective To set up the technique flat roof of nanogold lateral-flow-immunoassay for our department. Methods Nanogold colloid was prepared with a reduction of chloroauric acid by sodium citrate. Optimal conditions of pH and antibody concentration for the coating can be determined by comparing the absorption between 520 and 580 nm（A520－A580）(Eg. a curve was made with the value of （A520－A580）y-axis and the pH and antibody concentration x-axis respectively. The biocojugation of buffer solution was selected according to the level of aggregation. The proper pyroxylin membrane and glass cellulose membrane and the pretreatment solution of the conjugation pad and sample pad along with the desiccation conditions of the membranes were choosed according to the releasing speed and degree of the conjugated nano-ptarticles. Strips were made and the positive results agreement of detecting results was compared with that of the ELISA kit. Results 40nm nanoparticles were made successfully. The proper pH and antibody concentration and the conjugation buffer solution of antibody I were 8.5 and 78(g/ml nanogold and 10mM Tris-HCl (pH8.5) respectively. The proper pyroxylin membrane was whatman Immunopore RP. The glass cellulose membrane Ahlstrom8964 was selected as conjugation pad and sample pad. The pret