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J. Biol. Chem., Vol. 277, Issue 23, 20468-20476, June 7, 2002

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Characterization of Oligopeptides That Cross-react with Carbohydrate-specific Antibodies by Real Time Kinetics, In-solution Competition Enzyme-linked Immunosorbent Assay, and Immunological Analyses^*

Paul J. Brett, Harmale Tiwana, Ian M. Feavers^§, and Bambos M. Charalambous^¶

From the  Department of Medical Microbiology, Royal Free and University College Medical School, Royal Free Campus, University College London, Rowland Hill Street, London NW3 2PF and ^§ Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Hertfordshire EN6 3QG, United Kingdom

Phage displaying random cyclic 7-mer, and linear 7-mer and 12-mer peptides at the N terminus of the coat protein, pIII, were panned with the murine monoclonal antibody, 9-2-L379 specific for meningococcal lipo-oligosaccharide. Five cyclic peptides with two sequence motifs, six linear 7-mers, and five linear 12-mers with different sequence motifs were identified. Only phage displaying cyclic peptides were specifically captured by and were antigenic for 9-2-L379. Monoclonal antibody 9-2-L379 exhibited "apparent" binding affinities to the cyclic peptides between 11 and 184 nM, comparable with lipo-oligosaccharide. All cyclic peptides competed with the binding of 9-2-L379 to lipo-oligosaccharide with EC₅₀ values in the range 10-105 µM, which correlated with their apparent binding affinities. Structural modifications of the cyclic peptides eliminated their ability to bind and compete with monoclonal antibody 9-2-L379. Mice (C3H/HeN) immunized with the cyclic peptide with optimal apparent binding affinity and EC₅₀ of competition elicited cross-reactive antibodies to meningococcal lipo-oligosaccharide with end point dilution serum antibody titers of 3200. Cyclic peptides were converted to T-cell-dependent immunogens without disrupting these properties by C-terminal biotinylation and complexing with NeutrAvidin®. The data indicate that constrained peptides can cross-react with a carbohydrate-specific antibody with greater specificity than linear peptides, and critical to this specificity is their structural conformation.

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