利用免疫亲和层析技术去除母乳中高丰度蛋白的研究

A novel strategy for subtracting high-abundance proteins in human milk by immunoaffinity chromatography

目的建立并评价一种去除母乳中高丰度蛋白的方法,使低丰度蛋白得到富集,为通过蛋白质组学技术发现母乳中的重要活性蛋白奠定基础。方法将母乳作为混合蛋白抗原免疫动物制备多克隆抗体,随后用此多克隆抗体通过免疫亲和层析技术去除母乳中的高丰度蛋白,采用免疫印迹法对免疫亲和层析的效果进行验证。结果母乳中的4种高丰度蛋白均得到一定程度的去除,建立了去除母乳中高丰度蛋白的新方法。结论通过混合抗原所制备的多克隆抗体可有效去除其中的高丰度抗原,并可使低丰度蛋白得到富集。这一方法也可用于提高血浆等样品中低丰度蛋白的检出效率。

Objective To establish and evaluate a novel method for subtracting high-abundance proteins in order to enrich low abundance proteins in human milk, with the aim of discovering important bioactive factors by proteomics approach in the future. Methods The whole proteins in human milk were used as immunogens to prepare polyclonal antibody, and the high-abundance proteins were subtracted by immunoaffinity chromatography. The effect was subsequently evaluated by immunoblotting. Results Anti-human milk polyclonal antibody was successfully prepared following routine method. After immunoaffinity chromatography analysis, four kinds of high-abundance proteins in human milk were removed. Conclusions These data indicated that the anti-human milk polyclonal antibodies prepared by whole protein samples were available for the removal of high-abundance proteins and enriching low abundance proteins of the samples. Accordingly, this method could also be used in detecting low abundance proteins in sample such as serum.