基于琼脂糖膜为载体的抗体芯片工作条件的优化

Optimizing the preparation condition of agarose-based antibody microarray

目的:优化以琼脂糖修饰的玻片为载体的抗体微阵列的制备方法,提高微阵列信噪比及检测灵敏度。方法:选取单核细胞趋化蛋白1(MCP-1)作为待检的靶蛋白,在琼脂糖膜上首先固定MCP-1捕获抗体,经过洗涤、封闭,然后依次加入标准抗原、生物素标记的检测抗体以及亲和素标记的cy3,分别孵育、洗涤后,激光共聚焦扫描仪扫描获取图像,分析各点的荧光强度,根据荧光强度确定捕获抗体的最佳固定时间、温度以及最适宜的洗涤缓冲液。结果:与4、37℃过夜固定相比,捕获抗体室温(25℃左右)过夜固定时,蛋白质在载体上的固定效率和反应活性最高;捕获抗体过夜固定比固定2 h获得更高的检测灵敏度;另外,通过比较相同pH值不同溶质缓冲液的洗涤效应,pH 7.4的缓冲液PBST可获得更优信号强度。结论:本研究优化了以琼脂糖修饰的玻片为载体的蛋白质微阵列的反应条件,提高了微阵列的检测灵敏度。

Objectve To test the feasibility of enhancing the assay sensitivity of antibody microarray based on agarose film through optimizing some basic experimental conditions. Methods Modified glass slides were robotically printed with capture antibodies against monocyte chemoattractant protein 1 （ MCP-1 ）, then dilutions of the cytokine were applied to the arrays and the protein was detected with biotin-labeled antibody coupled with Cy3-conjugated streptavidin. A laser confocal scanner was used to obtain the images and the signal intensity was analyzed subsequently. A series of factors in the production of antibody washing buffers. Results vernight under room microarrays were analyzed： the The system showed the highest temperature （ about 25 ℃ ） compared phate-buffered saline containing Tween 20 （ pH 7.4） was immobilization temperature and time of capture antibody and signal intensity when the capture antibody was immobilized overnight under room temperature（about 25℃ ） compared with immobilization overnight under 4 ℃ and 37 ℃. Phosphate-buffered saline containing Tween 20（pH 7.4） was demonstrated to be a much more favorable washing buffer forthis system compared with tris-buffered saline containing Tween 20 （pH 7.4）. Conclusion The optimized conditions have been established to enhance the signal intensity of antibody microarray on the agarose film coated glass slide.