Affinity purification and characterization of an anti-PEG IgM
Affinity purification and characterization of an anti-PEG IgM
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DOI：
10.1002/jmr.920
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发表时间：
2009-03-01
影响因子：
2.7
通讯作者：
Malick, A. Waseem
中科院分区：
文献类型：
--
作者：
Ehrlich, George K.;Michel, Hanspeter;Malick, A. Waseem
以可变长度的聚乙二醇链(5、10、20和30 kDa)为亲和配基，用亲和层析法纯化抗-PEGIgM。用30 kDa的聚乙二醇化的NuGel(单代)观察到抗-PEGIgM的最大结合量。用表面等离子共振、Western blotting和ELISA法对纯化的抗-PEGIgM与聚乙二醇化功能蛋白/肽结合进行了鉴定。将20 kDa的聚乙二醇衍生物NuGel吸附于溶液中，经胃酶消化后进行亲和层析。对两种制剂的洗脱液进行了SDS-PAGE分析，得到一个大小相似的片段。然而，在溶液消化的亲和纯化材料中观察到一条额外的低分子量带，这是亲和基质上胃酶消化材料的洗脱液中没有的。较低的相对分子质量片段可以在较温和的条件下洗脱，表明失去了结合的多重性。通过质谱分析，得到各自片段的相对分子质量分别为132 kDa(两者)和82 kDa(溶液)。这两个片段的N-末端测序得到的初级序列(重链和轻链)不仅彼此相同，而且与天然IgM的序列相同。用ELISA法鉴定抗-PEGIgM片段与聚乙二醇化干扰素α-2a的结合。这些研究结果表明，亲和纯化的抗PEGIgM及其片段可以作为探针用于临床前研究和临床研究中的聚乙二醇化生物治疗药物的检测。版权所有(C)2008 John Wiley&Sons，Ltd.
Anti-PEG IgM was purified by affinity chromatography using variable length PEG chains (5, 10, 20 and 30 kDa) as affinity ligands. Maximal binding of anti-PEG IgM was observed using the 30 kDa PEG-derivatized NuGel (single passage). Purified anti-PEG IgM was characterized for binding to PEG functionalized proteins/peptides by surface plasmon resonance, western blotting and ELISA. Anti-PEG IgM, in solution and adsorbed on 20 kDa PEG-derivatized NuGel, was subjected to pepsin digestion followed by affinity chromatography. SDS-PAGE analysis of eluates in both preparations yielded one fragment that was similar in size. However, an additional lower molecular weight band was observed in solution-digested affinity purified material that was not present in the eluate from the material subjected to pepsin digestion on the affinity matrix. The lower MW fragment could be eluted under milder conditions, suggesting loss of binding multiplicity. Analysis by mass spectrometry yielded molecular weights of 132 kDa (both) and 82 kDa (solution) for the respective fragments. N-terminal sequencing of both fragments resulted in primary sequences (heavy and light chains) that were not only identical to each other but also to those of native IgM. The anti-PEG IgM fragments were characterized for binding to pegylated interferon alfa-2a by ELISA. The results from these studies suggest that affinity purified anti-PEG IgM and fragments can be used as probes in detection assays PEG functionalized biotherapeutics in pre-clinical and clinical studies. Copyright (c) 2008 John Wiley & Sons, Ltd.