Design of Adsorbents for Immunoaffinity Chromatography by Modification of Antibody

免疫亲和层析是一种高度纯化生物制品的优良方法，它依赖于抗原和抗体之间高度选择性和特异性的相互作用。在本研究中，通过对抗体配体进行化学修饰或切割，以提高其对蛋白水解消化的抵抗力或抑制免疫吸附剂的不良生理效应。用三聚氰尿酸活化聚乙二醇修饰抗体，并以修饰抗体为配体制备免疫吸附剂。这些免疫吸附剂对胰蛋白酶和胃蛋白酶具有较高的抗水解能力，对肽键水解位点具有特异性。对于没有特异性的pronase，它们也表现出比未修饰的免疫吸附剂更高的耐药性。在不保护抗体抗原结合位点的修饰下，随着修饰程度的增加，抗原的结合率降低，而有抗原结合位点的保护提高了结合率。由于PEG修饰提高了免疫吸附剂的抗蛋白水解消化能力，因此即使在含有蛋白酶的原料的免疫亲和层析中，修饰后的吸附剂也会显示出长条状。在体外循环中，通过免疫吸附剂去除免疫复合物，抗体配体可能引起不良的生理效应，如激活补体。由于一个抗体分子可以被木瓜蛋白酶切割成具有抗原结合位点的F_<ab>片段和具有抗体生理活性的F_c区域，因此使用F_<ab>片段作为配体可以避免这些不良影响。F_<ab>的吸附平衡与igg相似。pH和离子强度对F_<ab>和IgG的影响也相同。因此，F_<ab>片段可作为配体，以避免F_c区引起的不良生理影响。

Immunoaffinity chromatography, which depends on the highly selective and specific interaction between antigen and antibody, is an excellent method to purify bioproducts to high degrees. In the present work, antibody ligands were chemically modified or cleaved in order to improve the resistance to proteolytic digestion or to suppress undesirable physiological effects of immunoadsorbents.Antibodies were modified with polyethylene glycol activated with cyanuric chloride, and immunoadsorbents were prepared with use of modified antibodies as ligands. These immunoadsorbents showed the high resistance to proteolytic digestion by trypsin and pepsin, which have specificity for hydrolysis site of peptide bond. In the case of pronase, which has no specificity, they also showed higher resistance than unmodified immunoadsorbents. On modification without protection of antigen binding site of antibody, the binding ratio of antigen decreased with increase in the degree of modification, while protection of the site with antigen improved the binding ratio. Since the resistance to proteolytic digestion of immunoadsorbents is improved by PEG modification, the modified absorbents will show long lices even in immunoaffinity chromatography with crude material containing proteases.In extracorporeal circulation to remove immunocomplexes by immunoadsorbents, antibody ligands might cause undesirable physiological effects, such as activation of compliment. Since an antibody molecule can be cleaved by papain into F_<ab> fraction, which has antigen binding site, and F_c region, which shows physiological activities of antibody, these undesirable effects can be avoided by using F_<ab> fragment as ligand. The adsorption equilibrium of F_<ab> was similar to that of igg. The effects of pH and ionic strength were also the same in both F_<ab> and IgG. therefore, F_<ab> fragment is useful as ligand in order to avoid undersirable physiological effects caused by F_c region.