O'Donoghue G V, Okarma T B, Lee Y M
Department of Protein Biochemistry, Applied Immune Sciences Inc., Santa Clara, California 95054.
Anal Biochem. 1993 Sep;213(2):310-7. doi: 10.1006/abio.1993.1426.
A simple method to isolate immune complexes by column chromatography is described. The immune complex affinity column was constructed by the site-specific attachment of bovine conglutinin to agarose. Covalent attachment of conglutinin to agarose was achieved via hydrazone chemistry, which reacts aldehydes of oxidized oligosaccharides in the collagenous domain of conglutinin with hydrazide functional groups in the solid support. The constructed conglutinin affinity column captured complement-fixed model immune complexes of heat-aggregated human IgG and more classical complexes of chicken ovalbumin-anti-chicken ovalbumin. Neither the nonfixed immune complexes nor their individual components were retained by the column. The captured material was specifically eluted with EDTA without the use of low pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and protein sequence analysis of the eluates revealed the presence of the expected individual components, verifying that both antibody and antigen used to prepare the soluble immune complexes were recovered from the conglutinin column. The advantages of this approach over traditional methods of immune complex isolation and characterization are discussed.
本文描述了一种通过柱色谱法分离免疫复合物的简单方法。免疫复合物亲和柱是通过将牛凝集素位点特异性连接到琼脂糖上构建而成的。凝集素与琼脂糖的共价连接是通过腙化学实现的，该化学方法使凝集素胶原结构域中氧化寡糖的醛基与固体支持物中的酰肼官能团发生反应。构建的凝集素亲和柱捕获了热聚集人IgG的补体固定模型免疫复合物以及鸡卵清蛋白-抗鸡卵清蛋白的更典型复合物。未固定的免疫复合物及其单个组分均未被该柱保留。捕获的物质用EDTA特异性洗脱，无需使用低pH值。对洗脱液进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、蛋白质印迹分析和蛋白质序列分析，结果显示存在预期的单个组分，证实了用于制备可溶性免疫复合物的抗体和抗原均从凝集素柱中回收。讨论了该方法相对于传统免疫复合物分离和表征方法的优势。
