用ω-氨基己基琼脂糖阴性色谱法纯化人 IgG。
Purification of human IgG by negative chromatography on omega-aminohexyl-agarose.
机构信息
Department of Biotechnological Processes, School of Chemical Engineering, University of Campinas, UNICAMP, P.O. Box 6066, CEP 13083-970, Campinas, SP, Brazil.
出版信息
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Feb 15;878(5-6):557-66. doi: 10.1016/j.jchromb.2009.12.034. Epub 2010 Jan 4.
The omega-aminohexyl diamine immobilized as ligand on CNBr- and bisoxirane-activated agarose gel was evaluated for the purification of human immunoglobulin G (IgG) from serum and plasma by negative affinity chromatography. The effects of matrix activation, buffer system, and feedstream on recovery and purity of IgG were studied. A one-step purification process using Hepes buffer at pH 6.8 allowed a similar recovery (69-76%) of the loaded IgG in the nonretained fractions for both matrices, but the purity was higher for epoxy-activated gel (electrophoretically homogeneous protein with a 6.5-fold purification). The IgG and human serum albumin (HSA) adsorption equilibrium studies showed that the adsorption isotherms of IgG and HSA obeyed the Langmuir-Freundlich and Langmuir models, respectively. The binding capacity of HSA was high (210.4 mg mL(-1) of gel) and a positive cooperativity was observed for IgG binding. These results indicate that immobilizing omega-aminohexyl using bisoxirane as coupling agent is a useful strategy for rapid purification of IgG from human serum and plasma.
用 CNBr 和双环氧活化琼脂糖凝胶固定的ω-氨基己二胺作为配体，通过负亲和层析从血清和血浆中纯化人免疫球蛋白 G（IgG）。研究了基质活化、缓冲液体系和进料流对 IgG 回收率和纯度的影响。在 Hepes 缓冲液（pH 6.8）中进行的一步纯化过程，两种基质的负载 IgG 在未保留部分中的回收率相似（69-76%），但环氧活化凝胶的纯度更高（电泳均一的蛋白质，纯化倍数为 6.5 倍）。IgG 和人血清白蛋白（HSA）的吸附平衡研究表明，IgG 和 HSA 的吸附等温线分别符合 Langmuir-Freundlich 和 Langmuir 模型。HSA 的结合容量较高（凝胶 210.4mg/mL），并且观察到 IgG 结合的正协同性。这些结果表明，使用双环氧作为偶联剂固定ω-氨基己二胺是从人血清和血浆中快速纯化 IgG 的一种有效策略。
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