Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
J Immunol Methods. 2010 Jul 31;359(1-2):21-7. doi: 10.1016/j.jim.2010.05.003. Epub 2010 May 20.
A new electrophoretic technique for the qualitative and quantitative analyses of IgM isoforms and fragments has been developed. IgMs which are more complex than many other recombinantly expressed immunoglobulins are characterized by their high molecular weighted active forms and many additional isoforms and fragments in the molecular range between 25 and 1200kDa. To analyze the multimers, isoforms and fragments simultaneously a high-resolution method, which enables sufficient migration and separation is required. Furthermore, this method should be appropriate to analyze IgMs in crude culture supernatants as well as purified samples. Simple sample preparation avoiding unspecific protein loss has been established. Currently no standard method to analyze all of them accordingly is available. The IgM-SDS-PAGE investigated for this purpose includes all these aspects. The combination of simple sample preparation and the application of precast gels make this electrophoretic method suitable for research but also quality control. The selective quantification of the multimers and the relative isoform distribution were performed by sensitive Sypro Ruby staining obtaining reliable and reproducible data in clone screening and process development which has been demonstrated by recombinantly expressed IgMs with significantly different isoform pattern.
已经开发出一种用于 IgM 同种型和片段的定性和定量分析的新电泳技术。IgM 比许多其他重组表达的免疫球蛋白更为复杂，其特征是高分子量的活性形式以及在 25 至 1200kDa 分子量范围内的许多额外同种型和片段。为了同时分析多聚体、同种型和片段，需要一种能够进行充分迁移和分离的高分辨率方法。此外，该方法还应适用于分析粗培养上清液和纯化样品中的 IgM。已经建立了简单的样品制备方法，避免了非特异性蛋白质损失。目前，没有相应分析所有这些的标准方法。为此目的研究的 IgM-SDS-PAGE 包括了所有这些方面。简单的样品制备和预制凝胶的应用相结合，使这种电泳方法适用于研究和质量控制。通过敏感的 Sypro Ruby 染色进行多聚体的选择性定量和相对同种型分布，在克隆筛选和工艺开发中获得了可靠和可重复的数据，这已经通过具有显著不同同种型模式的重组表达 IgM 得到了证明。
