Mapping protein-protein contact sites using cellulose-bound peptide scans

We have characterized the interaction of two monoclonal antibodies with their respective antigens using cellulose-bound sets of overlapping peptides (peptide scans). Both antibodies CB/RS/5 and CB/MT/1 recognize discontinuous epitopes present in human interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-alpha). In addition, the interaction between TNF-alpha and its 55-kDa receptor (TNF-R) was investigated by the same approach. Both antibodies, as well as TNF-alpha, interacted with two or more regions of the peptide scans. Antibody-binding competition studies between the native antigens and peptides, covering single parts of the binding regions, enabled us to distinguish between binding to the paratope or other regions of the antibody. The combination of these experimental approaches allowed the identification of short antigen-derived sequences that are separated on the primary sequence but close in space on the surface of IL-10 and TNF-alpha, thus representing putative discontinuous epitopes. In the case of the TNF-R-derived peptide scans, two of the identified regions interact with the structurally similar TNF-beta in the TNF-beta-TNF-R complex. These data indicate that this approach should be generally applicable for mapping nonlinear protein-protein contact sites.