Fouraux Michael A, van der Heijden Annemarie, van Venrooij Walther J, Pruijn Ger J M
Department of Biochemistry, Nijmegen Center for Molecular Life Sciences, University of Nijmegen, the Netherlands.
Immunology. 2002 Jul;106(3):336-42. doi: 10.1046/j.1365-2567.2002.01432.x.
Coimmunoprecipitation studies with SW5, a frequently used and specific mouse monoclonal antibody (mAb) directed against the human La autoantigen, led to the identification of a functionally unrelated 80 000 MW protein, designated early endosome antigen 2 (EEA2). EEA2 appeared to be directly targeted by mAb SW5. Because an RNA-binding domain, a structural element of La containing the SW5-epitope, was not discernable in the primary structure of EEA2, the SW5-epitope on EEA2 was determined. Coiled-coil region 3 of EEA2 appeared to contain the epitope recognized by SW5. The SW5 epitope regions of La and EEA2 share a limited sequence homology and probably share a higher degree of structural similarity at the tertiary level. Most likely, the most critical determinants for recognition by SW5 reside in elements adopting alpha-helical conformations. These data indicate that the application of specific mAbs to purify and characterize (functionally) interacting proteins can be severely obscured by the cross-reactivity of mAbs with structurally, but not functionally, similar proteins.
使用SW5进行的共免疫沉淀研究，SW5是一种常用的针对人La自身抗原的特异性小鼠单克隆抗体（mAb），结果鉴定出一种功能上不相关的80000 MW蛋白质，命名为早期内体抗原2（EEA2）。EEA2似乎是mAb SW5的直接作用靶点。由于在EEA2的一级结构中未发现RNA结合结构域（La的一个包含SW5表位的结构元件），因此确定了EEA2上的SW5表位。EEA2的卷曲螺旋区域3似乎包含被SW5识别的表位。La和EEA2的SW5表位区域具有有限的序列同源性，并且在三级水平上可能具有更高程度的结构相似性。最有可能的是，SW5识别的最关键决定因素存在于采用α-螺旋构象的元件中。这些数据表明，特定mAb用于纯化和表征（功能上）相互作用的蛋白质时，可能会因mAb与结构相似但功能不相似的蛋白质发生交叉反应而受到严重干扰。
