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。以免疫纳米金(A520-A580)为纵坐标,相应的金标记参数(pH值、抗体量)为横坐标绘制曲线,曲线拐点为选择参数。标记缓冲体系的选择主要依据标记过程中纳米金聚沉与否确定。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和样品垫的玻璃纤维素膜、结合垫及样品垫预处理液配方。依据点加抗体的硝酸纤维素膜和结合垫在不同条件下干燥后组装试纸,免疫金的释放程度和速度确定干燥条件。组装试纸检测临床血清标本,比较与ELISA检测法的阳性符合率。结果成功制备了40 nm的纳米金。甲胎蛋白(AFP)抗体Ⅰ最适标记pH值为8.5,最适抗体标记量为78 mg/mL纳米金,标记缓冲体系为pH值8.5 10mM 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。结合垫选择pH值8.5的处理液预处理,样品垫选择pH值9.5的处理液预处理。硝酸纤维素膜封闭洗涤后在26℃湿度26%条件下干燥1 h,结合垫在26℃湿度26%条件下干燥2 h。当AFP含量大于10 ng/mL时,组装试纸检测临床血清标本的阳性检出率与ELISA法符合率为100%。结论成功建立了本组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。
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 p H and antibody concentration x-axis respectively. ）The biocojugation of buff er 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 Ⅰwere 8.5 and 78mg/ml nanogold and 10ram Tris HC1 （pH8. 5）respectively. The proper pyroxylin membrane was what man Immunopore RP. The glass cellulose membrane Ahlstrom8964 was selected as conjugation pad and sample pad. The pretreatment solution of conjugation pad and sample pad was pH 8.5 and pH9.5 respectively. After the pyroxylin membrane added with antibody Ⅱwas blocked and washed it was desiccated for lh under the condition of temperature 26 ℃ and humidity 26% while the conjugation pad added with immunogold was desiccated for 2h under the same conditions. When the content of AFP in serum was higher than 10ng/ml the positive accordance rate was 100 %. Conclusion A lateral-flow immunoassay poinPof care testing technique flat roof in our department is set up successfully, which could be used for the detection of infection of pathogenic bacteria and special marker of tumors.
出处
《国际检验医学杂志》
CAS
2011年第7期736-738,共3页
International Journal of Laboratory Medicine
基金
第三军医大学科技成果转化基金项目(2009XZH04)
关键词
甲胎蛋白类
纳米金
侧流免疫
试纸
技术平台
alpha fetoproteins
nanogold
lateral flow immunoassay
strip
technique flat roof
