Summary
The aim of this chapter is to provide a strategy for mapping linear antibody epitopes of protein antigens in order to discover candidates for vaccines or diagnostic tests. A set of overlapping peptides was designed and synthesised based upon a known amino acid sequence of the target protein, virulence-associated protein A (VapA) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals.
The peptides were biotinylated and used in an ELISA to screen immune sera from foals. These biotinylated peptides were coated directly onto micro titre plates that had been pre-coated with NeutrAvidin™. A linear B-cell epitope was identified by a universal recognition of sera to the synthetic peptides which corresponds to a particular fragment of the VapA protein.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Phumoonna, T., Barton, M. D., and Heuzenroeder, M. W. (2005) Recognition of a B-cell epitope of the VapA protein of Rhodococcus equi in newborn and experimentally infected foals. J. Vet. Med. B Infect. Dis. Vet. Public Health 52, 291–295.
Vanniasinkam, T., Barton, M. D., and Heuzenroeder, M. W. (2001) B-cell epitope mapping of the VapA protein of Rhodococcus equi: implications for early detection of R. equi disease in foals. J. Clin. Microbiol. 39, 1633–1637.
Panchanathan, V., Naidu, B. R., Devi, S., Pasqule, A. D., Mason, T., and Pang, T. (1998) Immunogenic epitopes of Salmonella typhi GroEL heat shock protein reactive with both monoclonal antibody and patients sera. Immunol. Lett. 62, 105–109.
Norton, R. E., Heuzenroeder, M., and Manning, P. A. (1996) Antigenic epitope mapping of the M24 protein of Streptococcus pyogenes: implications for serodiagnosis of rheumatic fever. FEMS Immunol. Med. Microbiol. 16, 267–271.
Torres, D., and Espino, A. M. (2006) Mapping of B-cell epitopes on a novel 11.5-kilodalton Fasciola hepatica–Schistosoma mansoni cross-reactive antigen belonging to a member of the F. hepatica saposin-like protein family. Infect. Immun. 74, 4932–4938.
Viswanathan, M., Anchin, J. M., Droupadi, P. R., Mandal, C., Linthicum, D. S., and Subramaniam, S. (1995) Structural predictions of the binding site architecture for monoclonal antibody NC6.8 using computer-aided molecular modeling, ligand binding, and spectroscopy. Biophys. J. 69, 741–753.
Sheriff, S., Silverton, E. W., Padlan, E. A., Cohen, G. H., Smith-Gill, S. J., Finzel, B. C., and Davies, D. R. (1987) Three-dimensional structure of an antibody–antigen complex. Proc. Natl. Acad. Sci. USA 84, 8075–8079.
Sekizaki, T., Takai, S., Egawa, Y., Ikeda, T., Ito, H., and Tsubaki, S. (1995) Sequence of the Rhodococcus equi gene encoding the virulence-associated 15–17-kDa antigens. Gene 155, 135–136.
Takai, S., Hines, S. A., Sekizaki, T., Nicholson, V. M., Alperin, D. A., Osaki, M., Takamatsu, D., Nakamura, M., Suzuki, K., Ogino, N., Kakuda, T., Dan, H., and Prescott, J. F. (2000) DNA sequence and comparison of virulence plasmids from Rhodococcus equi ATCC 33701 and 103. Infect. Immun. 68, 6840–6847.
Acknowledgements
We thank Stuart Rodda of Mimotopes for his advice and helpful discussion. We are grateful to Glenn Browning from University of Melbourne for kindly providing the sera. We also thank Tuck Weng Kok and the staff of the Serology Unit of the Institute of Medical and Veterinary Science for the use of equipment and facilities. This work was supported by the Rural Industries Research and Development Corporation (RIRDC) – Horse Programme and Vet Biotechnology Ltd, Adelaide, South Australia.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Heuzenroeder, M.W., Barton, M.D., Vanniasinkam, T., Phumoonna, T. (2009). Linear B-Cell Epitope Mapping Using Enzyme-Linked Immunosorbent Assay for Libraries of Overlapping Synthetic Peptides. In: Schutkowski, M., Reineke, U. (eds) Epitope Mapping Protocols. Methods in Molecular Biology™, vol 524. Humana Press. https://doi.org/10.1007/978-1-59745-450-6_10
Download citation
DOI: https://doi.org/10.1007/978-1-59745-450-6_10
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-934115-17-6
Online ISBN: 978-1-59745-450-6
eBook Packages: Springer Protocols