Friguet B, Fedorov A N, Djavadi-Ohaniance L
Unité de Biochimie Cellulaire (C.N.R.S. U.R.A. 1129), Institut Pasteur, Paris, France.
J Immunol Methods. 1993 Feb 3;158(2):243-9. doi: 10.1016/0022-1759(93)90220-2.
Gene expression of the beta subunit of E. coli tryptophan synthase in an E. coli cell-free transcription-translation system proceeds by pauses and produces a discrete but quite continuous pattern of nascent chains starting from the N terminus and ranging in size up to the 44 kDa end product corresponding to the completed beta chains. Using specific immunoadsorption of [35S]Met radiolabelled nascent chains by different monoclonal antibodies directed against the beta 2 subunit of E. coli tryptophan synthase, the size of the smallest N-terminal fragment reacting with each antibody has been determined by SDS electrophoretic analysis of the immunoadsorbed polypeptides. The immunoadsorption assay is performed in solution under conditions avoiding the usual drawbacks of solid phase immunoassay. This approach, in combination with the results obtained with a DNA fragment library permitted us to localize the antigenic determinants recognized by the monoclonal antibodies. The proposed method could help to localize rapidly the C-terminal boundary of an epitope, before starting systematic and precise mapping by other approaches. Moreover, the method described may be of general interest for the rapid production of a large set of C-terminal truncated polypeptides for studies of antigen-antibody recognition.
在大肠杆菌无细胞转录-翻译系统中，大肠杆菌色氨酸合酶β亚基的基因表达通过停顿进行，并产生从N端开始、大小范围直至对应完整β链的44 kDa终产物的离散但相当连续的新生链模式。利用针对大肠杆菌色氨酸合酶β2亚基的不同单克隆抗体对[35S]甲硫氨酸放射性标记的新生链进行特异性免疫吸附，通过对免疫吸附多肽的SDS电泳分析确定了与每种抗体反应的最小N端片段的大小。免疫吸附测定在溶液中进行，条件是避免固相免疫测定的常见缺点。这种方法与从DNA片段文库获得的结果相结合，使我们能够定位单克隆抗体识别的抗原决定簇。所提出的方法有助于在通过其他方法开始系统而精确的图谱绘制之前，快速定位表位的C端边界。此外，所描述的方法对于快速产生大量C端截短的多肽以研究抗原-抗体识别可能具有普遍意义。
