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的高度选择性和特异性相互作用。本工作通过对抗体配体进行化学修饰或裂解，以提高抗体的抗蛋白水解性或抑制免疫吸附剂的不良生理效应，用三聚氰酸活化的聚乙二醇改性抗体，并以修饰后的抗体为配体制备免疫吸附剂。这些免疫吸附材料对胰酶和胃酶的蛋白水解性表现出很高的抵抗力，这两种酶对肽键的水解点具有特异性。对于无特异性的链霉菌素酶，它们也表现出比未经修饰的免疫吸附材料更高的抗性。在不保护抗体抗原结合部位的修饰下，抗原结合率随修饰程度的增加而降低，而有抗原保护的部位则提高了结合率。由于聚乙二醇改性提高了免疫吸附剂的抗蛋白水解性，即使在与含有蛋白质的粗品亲和层析时，修饰后的吸附剂也会出现长条带。在体外循环中，通过免疫吸附剂去除免疫复合体，抗体配体可能会引起不良的生理效应，如对补体的激活。由于木瓜酶可将抗体分子切割成具有抗原结合部位的F_lt；ab&gt；片段和显示抗体生理活性的F_c区，因此使用F_lt；ab&gt；片段作为配基可以避免这些不良影响。F&lt；ab&Gt；的吸附平衡与Ig G的吸附平衡相似。PH和离子强度对F&lt；ab&Gt；和Ig G的影响也是相同的。因此，F&lt；ab&gt；片段可作为配基使用，以避免Fc区引起的不良生理效应。

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.