蛋白質微陣列已成為蛋白質體學研究中的重要工具。蛋白質微陣列的優勢在於可同時進行大量分析,但所需成本較高,故降低成本有其必要性。 本研究目的在於發展一低成本之冷光蛋白質微陣列之技術平台。以MicroCaster arrayer手動式打點機,將anti-human interleukin-2 polyclonal antibody及anti-human interleukin-4 polyclonal antibody點製於nitrocellulose membrane,以標定horseradish peroxidase的streptavidin結合帶有biotinylated anti-human interleukin-2及biotinylated anti-human interleukin-4 monoclonal antibody應用類似sandwich type之enzyme-linked immunosorbent assay反應,以偵測recombinant human interleukin-2及recombinant human interleukin-4。使用horseradish peroxidase產生之chemiluminescence以charge-coupled device拍攝,藉 由ScanAlyze軟體分析。 本技術平台以冷光基質反應產生chemiluminescence,由於產生發光 之反應中間物會向外擴散,故本系統最高點製密度為192點。本系統反 應條件確立後,recombinant human interleukin-4與recombinant human interleukin-2偵測極限分別為0.125 ng/ml和0.5 ng/ml,檢量線跨越2個 級數。 由於蛋白質微陣列缺乏類似polymerase chain reaction的反應提高靈敏度,故應用streptavidin-peroxidase polymer與streptavidin-biotin complex兩種方式,藉由提高反應訊號,以求更低的偵測極限。結果顯示雖然有 使蛋白質微陣列之偵測信號增加,但未降低偵測極限。
A low-cost, low-density protein microarray on nitrocellulose membrane for the detection of human cytokines was developed. The detection was based on chemiluminescent sandwich-type immunoassay. The printed spots were uniform and consistent in size. Under optimized condition, the limits of detection for human interleukin 2 and interleukin 4 were 0.5 and 0.125 ng/ml, respectively. The linear range of the calibration curves spanned over two orders of magnitude. The effects of several potential signal enhancers were also tested.