Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria.
J Chromatogr A. 2011 Apr 29;1218(17):2374-80. doi: 10.1016/j.chroma.2010.10.053. Epub 2010 Oct 21.
We have developed a method for quantification of a specific monoclonal IgM directed toward embryonic stem cells based on a peptide affinity monolith. A peptide affinity ligand with the sequence C-C-H-Q-R-L-S-Q-R-K was obtained by epitope mapping using peptide SPOT synthesis. The peptide ligand was covalently immobilized by coupling the N-terminal cysteine to a monolithic disk that was previously modified with iodated spacer molecules. The monolithic disc was used for quantification of purified IgM and for IgM present in mammalian cell culture supernatant. We observed 17% unspecific binding of IgM to the monolithic disk and additionally a product loss in the flow through of 20%. Nevertheless, calibration curves had high correlation coefficients and inter/intra-assay variability experiments proved sufficient precision of the method. A limit of quantification of 51.69 μg/mL for purified IgM and 48.40 μg/mL for IgM in cell culture supernatant could be calculated. The binding capacity was consistent within the period of the study which included more than 200 cycles. The analysis time of less than 2 min is an advantage over existing chromatographic methods that rely on pore diffusion.
我们开发了一种基于肽亲和整体柱的方法来定量特定的针对胚胎干细胞的单克隆 IgM。通过使用肽 SPOT 合成进行表位作图，获得了序列为 C-C-H-Q-R-L-S-Q-R-K 的肽亲和配体。肽配体通过将 N 端半胱氨酸偶联到先前用碘代间隔分子修饰的整体盘中进行共价固定。该整体盘用于定量纯化的 IgM 和哺乳动物细胞培养上清液中的 IgM。我们观察到 17%的 IgM 非特异性结合到整体盘上，并且在流通过程中还损失了 20%的产物。尽管如此，校准曲线仍具有高相关系数，并且日内/日间精密度实验证明该方法具有足够的精度。可以计算出纯化的 IgM 的定量下限为 51.69 μg/mL，细胞培养上清液中的 IgM 为 48.40 μg/mL。在包括 200 多个循环的研究期间，结合容量保持一致。分析时间不到 2 分钟，这是优于依赖孔扩散的现有色谱方法的优势。
