Antibody immobilization to high-performance liquid chromatography supports - Characterization of maximum loading capacity for intact immunoglobulin G and Fab fragments
Antibody immobilization to high-performance liquid chromatography supports - Characterization of maximum loading capacity for intact immunoglobulin G and Fab fragments
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DOI：
10.1016/s0021-9673(00)00548-3
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发表时间：
2000-08-04
影响因子：
4.1
通讯作者：
Hage, DS
中科院分区：
文献类型：
--
作者：
Clarke, W;Beckwith, JD;Hage, DS
本研究检查了影响完整免疫球蛋白G（IgG）或Fab片段的最大量的各种因素，这些片段可以共价固定到硅胶和其他HPLC级支持物上，用于免疫亲和色谱或免疫提取。考虑的因素包括可用于固定的表面积、载体的孔径、固定方法的类型和载体基质的性质。发现确定固定化程度的主要因素是支持物的表面积与IgG或Fab片段到达该表面的能力之间的关系。进入支持物表面是被固定的蛋白质的大小和支持物孔隙率的函数，其中最大固定是用具有对于完整IgG约300埃和对于Fab片段约100埃的孔径的支持物获得的。在不同的偶联方法之间注意到固定化的最大水平的一些差异。当在具有可比孔径的材料之间进行比较时，像波罗斯和Emphaze这样的载体给出了与用HPLC级二氧化硅观察到的结果相似的结果。在这项工作中观察到的IgG和Fab片段的许多趋势应该适用于其他蛋白质，将被固定到HPLC支持。(C)2000 Elsevier Science B.V.保留所有权利。
This study examined various factors that affect the maximum amount of intact immunoglobulin G (IgG) or Fab fragments that can be covalently immobilized to silica and other HPLC-grade supports for use in immunoaffinity chromatography or immunoextractions. Factors that were considered included the amount of surface area available for immobilization, the pore size of the support, the type of immobilization method and the nature of the support matrix. The main factor in determining the extent of immobilization was found to be the relationship between the support's surface area and the ability of the IgG or Fab fragments to reach this surface. Access to the support surface was a function of the size of the protein being immobilized and the support porosity, with maximum immobilization being obtained with supports having pore sizes of approximately 300 Angstrom for intact IgG and 100 Angstrom for Fab fragments. Some differences in the maximum level of immobilization were noted between different coupling methods. Supports like Poros and Emphaze gave similar results to those seen with HPLC-grade silica when a comparison was made between materials with comparable pore sizes. Many of the trends observed in this work for IgG and Fab fragments should apply to other proteins that are to be immobilized to HPLC supports. (C) 2000 Elsevier Science B.V. All rights reserved.