Requirement of multiple phage displayed peptide libraries for optimal mapping of a conformational antibody epitope on CCR5

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• 作者：O'Connor ; Karen H. ; K脙脙nigs ; Christoph ; Rowley ; Merrill J. ; Irving ; James A. ; Wijeyewickrema ; Lakshmi C. ; et. al.
• 关键词：Epitope ; Phage library ; Chemokine receptor ; CCR5 ; HIV infectivity ; Type II collagen
• 刊名：Journal of Immunological Methods
• 出版年：2005
• 期刊代码：57_00221759
• 类别：imm
• 出版时间：April, 2005
• 卷：299
• 期：1-2
• 页码：21-35
• 文件大小：371 K

摘要

In the absence of information from crystallography, conformational epitopes can often be discerned by antibody screening of phage displayed random peptide libraries. However the context in which the peptide is displayed, and the number of copies displayed in the library, can influence results and interpretations. Here, the monoclonal antibodies 3A9 specific for the transmembrane chemokine receptor CCR5, and CII-C1 specific for type II collagen, were used to screen multiple phage-displayed peptide libraries in which peptides were displayed in either the pIII or pVIII coat proteins. ELISA was used to test for reactivity and cross-inhibitory activity of isolated phage clones. Based on sequences of reactive phage inserts, epitope motifs were initially inferred from a molecular model of CCR5 and subsequently confirmed experimentally using mutagenesis to alanine. For each mAb, phage sequences from pIII biopannings were more diverse than from pVIII biopannings. Notably, sequences from either biopanning were cross-inhibitory despite a lack of linear sequence homology. For CCR5, residues ⁸⁸H and ⁹⁴W in the first loop of CCR5 were identified by pIII biopannings, and ⁷S⁹IYD¹¹ at the N-terminus by pVIII biopannings. Thus conformational epitopes can be identified using phage display, but optimal mapping of complex epitopes can require the use of multiple peptide libraries.