Fack F, Hügle-Dörr B, Song D, Queitsch I, Petersen G, Bautz E K
Institute of Molecular Genetics, University Heidelberg, Germany.
J Immunol Methods. 1997 Aug 7;206(1-2):43-52. doi: 10.1016/s0022-1759(97)00083-5.
We present a comparative study on epitope mapping of four monoclonal antibodies directed against four different antigens using alternative phage display techniques and peptide scanning: mAb215 reacts with the largest subunit of RNA polymerase II, mAbBp53-11 with the tumor suppressor protein p53, mAbGDO5 with the Hantaan virus glycoprotein G2 and mAbL13F3 with the Hantaan virus nucleocapsid protein. Epitopes were determined (i) by gene-fragment phage display libraries, constructed by DNaseI digested random gene fragments cloned into the 5' terminus of the pIII-gene of fd phage and (ii) by random peptide phage libraries displaying 6mer and 15mer peptides at the N-terminus of the pIII protein. Using the gene-fragment phage display libraries a single round of affinity selection resulted in the determination of the corresponding epitopes for all monoclonal antibodies tested. In contrast, biopanning of 6mer and 15mer random peptide libraries was only successful for two of the antibodies (mAbBp53-11 and mAbGDO5) after three or four rounds of selection. For the anti-p53 antibody we recovered the epitope from both the 6mer and 15mer library, for mAbGDO5 only the 6mer library displayed the epitope sequence. However, screening of the random peptide libraries with mAb215 and mAbL13F3 failed to yield immunopositive clones. Fine mapping of the epitopes by peptide scan revealed that the minimal epitopes recognized by mAbBp53-11 and mAbGDO5 consist of four and five amino acids, respectively, whereas mAb215 requires a minimal epitope of 11 amino acids for antigen recognition. In contrast, mAbL13F3 did not react with any of the synthesized 15mer peptides. The limits of the different methods of epitope mapping tested in this study are compared and the advantages of the gene-fragment phage display system are discussed.
单克隆抗体215与RNA聚合酶II的最大亚基反应，单克隆抗体Bp53-11与肿瘤抑制蛋白p53反应，单克隆抗体GDO5与汉坦病毒糖蛋白G2反应，单克隆抗体L13F3与汉坦病毒核衣壳蛋白反应。表位通过以下方法确定：(i) 基因片段噬菌体展示文库，由经DNaseI消化的随机基因片段构建，这些片段克隆到fd噬菌体pIII基因的5'末端；(ii) 随机肽噬菌体文库，在pIII蛋白的N末端展示6聚体和15聚体肽。使用基因片段噬菌体展示文库，一轮亲和选择就确定了所有测试单克隆抗体的相应表位。相比之下，6聚体和15聚体随机肽文库的生物淘选在经过三轮或四轮选择后仅对两种抗体（单克隆抗体Bp53-11和单克隆抗体GDO5）成功。对于抗p53抗体，我们从6聚体和15聚体文库中都回收了表位，对于单克隆抗体GDO5，只有6聚体文库展示了表位序列。然而，用单克隆抗体215和单克隆抗体L13F3筛选随机肽文库未能产生免疫阳性克隆。通过肽扫描对表位进行精细作图表明，单克隆抗体Bp53-11和单克隆抗体GDO5识别的最小表位分别由四个和五个氨基酸组成，而单克隆抗体215识别抗原需要11个氨基酸的最小表位。相比之下，单克隆抗体L13F3与任何合成的15聚体肽都不反应。比较了本研究中测试的不同表位作图方法的局限性，并讨论了基因片段噬菌体展示系统的优点。
