Johansen K, Svensson L
Department of Virology, Swedish Institute for Infectious Diseases Control, Stockholm, Sweden.
Methods Mol Med. 1998;13:15-28. doi: 10.1385/0-89603-485-2:15.
Immunoprecipitation allows the investigator to detect and quantitate antigens in a mixture of proteins or characterize a specific antibody response to already well-characterized proteins. Addition of antibodies to proteins, usually radiolabeled, allows formation of antigen-antibody complexes. After separation from contaminating proteins, the complexes are disassociated and the proteins of interest are separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Size and quantity of proteins may then be analyzed either by autoradiography or a gel scanning procedure. Immunoprecipitation is extremely sensitive and may detect very small amounts of radiolabeled protein (detection level ~100 pg protein or 100 cpm/protein). Unlabeled proteins may be used if other sensitive detection methods are utilized, e.g., enzymatic activity assays or Western blotting. The advantage of the immunoprecipitation technique vs immunoblotting is the possibility to analyze the immune response to proteins expressed in their native conformation. Radioimmunoprecipitation assay (RIPA) is used routinely for the detection of viral proteins, characterization of monoclonal and polyclonal antibody preparations, and determination of the specificity of the immune response to various pathogens (1-3).
免疫沉淀法可使研究人员检测和定量蛋白质混合物中的抗原，或表征针对已充分表征的蛋白质的特异性抗体反应。向通常为放射性标记的蛋白质中添加抗体，可形成抗原-抗体复合物。从污染蛋白中分离后，复合物解离，感兴趣的蛋白质通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行分离。然后可通过放射自显影或凝胶扫描程序分析蛋白质的大小和数量。免疫沉淀法极其灵敏，可检测到极少量的放射性标记蛋白质（检测水平约为100 pg蛋白质或100 cpm/蛋白质）。如果使用其他灵敏的检测方法，如酶活性测定或蛋白质印迹法，则可使用未标记的蛋白质。免疫沉淀技术相对于免疫印迹法的优势在于能够分析针对天然构象表达的蛋白质的免疫反应。放射免疫沉淀测定法（RIPA）通常用于检测病毒蛋白、表征单克隆和多克隆抗体制剂，以及确定对各种病原体的免疫反应特异性（1-3）。
