A method for high-throughput, sensitive analysis of IgG Fc and Fab glycosylation by capillary electrophoresis
A method for high-throughput, sensitive analysis of IgG Fc and Fab glycosylation by capillary electrophoresis
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DOI：
10.1016/j.jim.2014.12.004
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发表时间：
2015-02-01
影响因子：
2.2
通讯作者：
Alter, Galit
中科院分区：
文献类型：
--
作者：
Mahan, Alison E.;Tedesco, Jacquelynne;Alter, Galit
IgG恒定区（Fc）的N-聚糖在体内调节和指导多种抗体功能（包括抗体依赖性细胞毒性、补体沉积和炎症调节等）中发挥核心作用。然而，包括HPLC和质谱法的N聚糖分析的传统方法在技术上具有挑战性，并且不适合处理在多克隆IgG的N-糖基化的临床或动物研究中分析的大量低浓度样品。在这里，我们描述了一种基于毛细管电泳的技术，以快速准确地分析血浆来源的多克隆IgG-糖基化，具有成本效益，灵敏的方式，非常适合高通量分析。此外，由于多克隆IgG的显著部分在Fc和Fab结构域上糖基化，我们开发了一种分离和分析多克隆人、恒河猴和小鼠IgG中的结构域特异性糖基化的方法。总的来说，该方案允许快速、准确和灵敏地分析Fc特异性IgG糖基化，这对于在人群水平研究抗体糖基化如何响应疫苗接种或感染以及在临床和动物研究中从自身免疫到癌症的疾病状态之间变化至关重要。(C))2014爱思唯尔有限公司版权所有。
The N-glycan of the IgG constant region (Fc) plays a central role in tuning and directing multiple antibody functions in vivo, including antibody-dependent cellular cytotoxicity, complement deposition, and the regulation of inflammation, among others. However, traditional methods of Nglycan analysis, including HPLC and mass spectrometry, are technically challenging and ill suited to handle the large numbers of low concentration samples analyzed in clinical or animal studies of the N-glycosylation of polyclonal IgG. Here we describe a capillary electrophoresis-based technique to analyze plasma-derived polyclonal IgG-glycosylation quickly and accurately in a cost-effective, sensitive manner that is well suited for high-throughput analyses. Additionally, because a significant fraction of polyclonal IgG is glycosylated on both Fc and Fab domains, we developed an approach to separate and analyze domain-specific glycosylation in polyclonal human, rhesus and mouse IgGs. Overall, this protocol allows for the rapid, accurate, and sensitive analysis of Fc-specific IgG glycosylation, which is critical for population-level studies of how antibody glycosylation may vary in response to vaccination or infection, and across disease states ranging from autoimmunity to cancer in both clinical and animal studies. (C)) 2014 Elsevier B.V. All rights reserved.