蛋白芯片标记系统的优化研究  

Optimization of protein microarray marking system

作　　者：罗进[1] 李丁[1] 张文红[1] 王琰[1] 杨浩[1] 朱文伟[1] 夏琳[1] 韩锋产[1] 阎小君[1] 

机构地区：[1]第四军医大学药学系全军基因诊断研究所,陕西西安710033

出　　处：《第四军医大学学报》2005年第4期311-313,共3页Journal of the Fourth Military Medical University

摘　　要：目的:探讨一种能够更好的制备单一均匀的纳米 免疫金颗粒的方法,使蛋白芯片在临床诊断疾病时更为灵敏、 准确.方法:利用不同的方法制备3种胶体金,用分光光度计 对其均一性和颗粒大小进行评价;用麦氏方法寻找出3种胶 体金的最佳蛋白标记量;根据免疫金的制备条件设计5种离 心条件,制备出15种免疫金;利用质控蛋白芯片、梅毒检测蛋 白芯片和弓形虫检测芯片分别对15种免疫金显色,通过 XK2100阅读仪得到其相应的灰度值,分析这些数值来确定免 疫金的稳定性和灵敏度,最终确定相对优化的免疫金及其制 备工艺.结果:在制备200mL胶体金的3种配方中以400g/L 氯金酸50μL与10g/L的柠檬酸三钠溶液8mL得到的胶体 金颗粒较为均匀;而该胶体金以30100g,离心2次得到的免 疫金能够在灵敏度和稳定性之间达到一个较好的平衡点. 结论:改进后的免疫金制备方法是更优良的方案.AIM: To develop a better method to prepare an uniform immunogold to promote the sensitivity and stability of microarray in the diagnosis of diseases.METHODS: Colloidal gold was prepared by three methods and the products were analyzed by spectrophotometry.The optimized amount of staphylococcal protein A (SPA) was determined by Mey experiment.The fixed amount of SPA was labelled to the colloidal gold and the immunogold was purified under five different centrifugal conditions.Fifteen kinds of immunogolds were obtained.IgG,Toxoplasma (Tox) antigen and Treponema palidum (TP) antigen were dotted on the nitrocellulose filter membrane and proteinarrays were made up. XK2100 reader was used to test the colour of the fifteen kinds of immunogolds by standard microarray,Tox proteinarray and TP proteinarray.The data were analyzed for the relatively optimal method.RESULTS: Among the three methods,the colloidal gold made by adding 8 mL of 10 g/L citrated sodium to 50 μL of 400 g/L chloroauric acid was more stable and more suitable for proteinarray.The immunogold derived from this colloidal gold centrifuged twice at 14,000 rpm was sensitive and stable.CONCLUSION: Our improved method is optimal for the preparation of immunogold.

关 键 词：胶体金 免疫金 微阵列 

分 类 号：R446.62[医药卫生—诊断学]