Luo Quanzhou, Zou Hanfa, Zhang Qiang, Xiao Xiangzhu, Ni Jianyi
National Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China.
Biotechnol Bioeng. 2002 Dec 5;80(5):481-9. doi: 10.1002/bit.10391.
Reactive monoliths of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) have been prepared by "in-situ" copolymerization of the monomers in the presence of porogenic diluents. Protein A and L-histidine were immobilized on the monoliths directly or through a spacer arm, respectively. The properties of these two kinds of affinity columns were characterized, and the results showed that the columns with coupling of ligands by a spacer arm have some extent of non-specific adsorption for bovine serum albumin. The affinity column based on the monolithic polymer support provided us with good hydrodynamic characteristic, low flow resistance, and easy preparation. These two affinity columns were used for the purification of immunoglobulin G from human serum. The purity of the purified IgG was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The stability of the protein A affinity column was investigated, and its performance remained invariable after half a year. The effects of the nature and the pH of the buffer system on the adsorption capacity of human IgG on histidyl affinity column were also investigated. The protein A affinity column is favorable for rapid analysis of human IgG samples. In contrast, the advantages of mild elution conditions, high stability, as well as low cost provide the histidyl column further potential possibility for fast removal of IgG from human plasma in clinical applications.
通过在致孔稀释剂存在下使单体“原位”共聚，制备了大孔聚（甲基丙烯酸缩水甘油酯 - 二甲基丙烯酸乙二醇酯）的反应性整体柱。蛋白A和L - 组氨酸分别直接或通过间隔臂固定在整体柱上。对这两种亲和柱的性能进行了表征，结果表明通过间隔臂偶联配体的柱子对牛血清白蛋白有一定程度的非特异性吸附。基于整体聚合物载体的亲和柱具有良好的流体动力学特性、低流动阻力且易于制备。这两种亲和柱用于从人血清中纯化免疫球蛋白G。通过基质辅助激光解吸电离飞行时间质谱（MALDI - TOF - MS）检测纯化的IgG的纯度。研究了蛋白A亲和柱的稳定性，半年后其性能保持不变。还研究了缓冲系统的性质和pH对人IgG在组氨酸亲和柱上吸附容量的影响。蛋白A亲和柱有利于人IgG样品的快速分析。相比之下，温和的洗脱条件、高稳定性以及低成本等优点为组氨酸柱在临床应用中从人血浆中快速去除IgG提供了进一步的潜在可能性。
