Burd R S, Raymond C S, Ratz C A, Dunn D L
Department of Surgery, University of Minnesota, Minneapolis 55455.
Hybridoma. 1993 Feb;12(1):135-42. doi: 10.1089/hyb.1993.12.135.
A method for purifying IgM monoclonal antibodies (mAbs) from murine ascites using a DEAE-disk is described. After ammonium sulfate precipitation, ascites proteins are redissolved and loaded onto a DEAE-disk. MAb then is eluted from the disk using a stepwise NaCl gradient. IgM mAb produced by this procedure was > 95% pure as assessed by reducing SDS-PAGE analysis and was free of significant IgG contamination as determined by double radial immunodiffusion analysis. Yield of IgM mAb was approximately 2% of total ascites protein and approximately 10% of the amount of IgM contained in crude ascites. MAb retained immunoreactivity as assessed by ELISA, and the affinity index was evaluated by thiocyanate elution and remained unchanged. This two step technique for purifying IgM mAb from murine ascites is rapid, simple, and yields mAb of sufficient purity and immunoreactivity for the majority of mAb applications.
描述了一种使用DEAE圆盘从鼠腹水纯化IgM单克隆抗体（mAb）的方法。硫酸铵沉淀后，腹水蛋白重新溶解并加载到DEAE圆盘上。然后使用逐步NaCl梯度从圆盘上洗脱mAb。通过还原SDS-PAGE分析评估，该方法产生的IgM mAb纯度>95%，通过双径向免疫扩散分析确定无明显IgG污染。IgM mAb的产量约为腹水总蛋白的2%，约为粗腹水所含IgM量的10%。通过ELISA评估，mAb保留了免疫反应性，并通过硫氰酸盐洗脱评估了亲和指数，其保持不变。这种从鼠腹水纯化IgM mAb的两步技术快速、简单，产生的mAb纯度和免疫反应性足以满足大多数mAb应用。
