Beyer Natascha Helena, Schou Christian, Houen Gunnar, Heegaard Niels H H
Department of Autoimmunology, Statens Serum Institut, Artillerivej 5, Copenhagen S, Denmark.
J Immunol Methods. 2008 Jan 31;330(1-2):24-33. doi: 10.1016/j.jim.2007.10.016. Epub 2007 Nov 21.
A method for the identification of protein antigens captured in electroimmunoprecipitates was developed. Different antigen-antibody precipitates were generated by agarose gel immunoelectrophoresis. The immunoprecipitates were excised and various methods for extracting and dissociating the precipitates were systematically studied by analyzing for protein components of the extracts using peptide mass fingerprinting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal recovery of antigen was obtained by 24-h extraction at 37 degrees C using a minimal volume of 0.06 M Tris-HCl, 10% SDS (pH 7). This simple and robust method is useful for the characterization of antibody specificity. It can also be used to identify antigens generating unknown precipitates in crossed immunoelectrophoresis with polyspecific antisera, including human IgG-antigen complexes electroimmunoprecipitated by secondary antibodies. Thus, the method may prove useful as an additional technique in biomarker discovery.
开发了一种用于鉴定电免疫沉淀中捕获的蛋白质抗原的方法。通过琼脂糖凝胶免疫电泳产生不同的抗原-抗体沉淀物。切除免疫沉淀物，并通过在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后使用肽质量指纹图谱分析提取物的蛋白质成分，系统地研究了提取和解离沉淀物的各种方法。使用最小体积的0.06 M Tris-HCl、10% SDS（pH 7）在37℃下提取24小时可获得最佳的抗原回收率。这种简单且可靠的方法对于表征抗体特异性很有用。它还可用于鉴定在与多特异性抗血清的交叉免疫电泳中产生未知沉淀物的抗原，包括由二抗电免疫沉淀的人IgG-抗原复合物。因此，该方法可能被证明是生物标志物发现中的一种有用的附加技术。
