Optimization of protein microarray marking system

    LUO Jin,LI Ding,ZHANG Wenª²Hong,WANG Yan,YANG Hao,ZHU Wenª²Wei,XIA Lin,HAN Fengª²Chan,YAN Xiaoª²Jun

    PLA Institute of Genetic Diagnosis,School of Pharmacology,Fourth Military Medical University,Xi¬ðan 710033,China

    ¡¾Abstract¡¿ 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 ¦ÌL 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. ......