Study On New Technology Of High Throughput Protein Microarray And Its Applications

With the development of the research of proteomics, there are increasing needs in protein measurement including protein profiling and protein function research. Protein microarrays have shown their encouraging prospect in protein analysis due to their powerful potential such as high parallel and high sensitivity.In this paper, we described three novel methods of protein microarray including high throughput antibody screening, sensitive detection method based on silver enhancement and microcontact protein printing, respectively. First, we developed a novel high throughput antibody screening method based on an antigen microarray technique to assay multiplex targets. This novel assembly protein microarray combines the advantages of microplate and protein microarray. The protein microarray was consisted of six slides and contains 108 reaction wells, in which there is a submicroarray containing 100 protein spots. In our experiment, antigen microarrays with 35 antigens in each reaction well were fabricated and then their analytical capability were checked by fluorescent method such as reproducibility of intra-slide and inter-slides, homogeneity of the duplicated spots, the lowest detectable concentration and linear range of target detection. In addition, the sensitivity and linear range between this method and ELISA were compared. Then 75 mouse antibodies with regard to 35 antigens were assayed. Their affinities and specificities were obtained simultaneously based on their fluorescent signals. The present method shows its advantages such as flexibility, high throughput and high sensitivity. It has great potential to be used in the processes of large scale production of antibodies.In the second part, an antibody microarray for simultaneous detection of AFP and CEA was fabricated, in which a detection method based on nano-gold immunological amplification coupled with silver enhancement was developed. We optimized the time of various silver enhancement and obtained a typical dose-response curve of antigens with antibody array. The limit of detection was 5.1ng/ml for AFP and 4.0ng/ml for CEA rspectively. It showed good performance in assay of two tumor markers in the quality control serum. The results of silver enhancement can be visualized by naked eye. As a result, the presented method is suitable for primary diagnosis in the large scale physical examination.In the last part, we developed a set of method of microarray's fabrication and detection, which were consisted of the agarose stamper and the silver enhancement method respectively. We evaluated the ability of agarose stamper in repetitive stamping. Then direct and sandwich strategies were employed to perform quantitative analysis using the protein array based on this conjugation technique, respectively. The limits of detection are 0.33fmol for direct direction and 0.13fmol for sandwich detection. This integration technique has its promising to be a set of routine protein array technique due to its cost-effective and versatile fabrication and sensitive detection.