Abstract
Peptides (8–20 residues) are as effective as proteins in raising antibodies, both polyclonal and monoclonal with a titer above 20,000 easily achievable. A successful antipeptide antibody production depends on several factors such as peptide sequence selection, peptide synthesis, peptide–carrier protein conjugation, the choice of the host animal, and antibody purification. Peptide sequence selection is likely the most difficult and critical step in the development of antipeptide antibodies. Although the format for designing peptide antigens is not precise, several guidelines can help maximize the likelihood of producing high-quality antipeptide antibodies. Typically, 5–20 mg of peptide is enough for raising an antibody, for preparing a peptide affinity column, and for antibody titer determination using an enzyme-linked immunosorbent assay (ELISA). Usually, it takes 3 months to raise a polyclonal antipeptide antibody from a rabbit that yields ~90 mL of serum which translates into approximately 8–10 mg of the specific antibody after peptide affinity purification.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Howard, G. C. and Kaser, M. R., eds. (2007) Making and Using Antibodies: A Practical Handbook. CRC Press, Taylor & Francis, Boca Raton, FL.
Harlow, D. L. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, New York.
Rosenberg, I. M. (1996) Protein Analysis and Purification Benchtop Techniques. Birkhauser, Boston, MA.
Walker, J. M., ed. (1996) The Protein Protocols Handbook. Humana Press, Totowa, NJ.
Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober, W. (1996) Current Protocols in Immunology. John Wiley & Sons, New York, vol. 2, 9.0.1–9.8.15.
Delves, P. J. (1997) Antibody Production Essential Techniques. John Wiley & Sons, New York.
Tsigelny, I. F., ed. (2002) Protein Structure Prediction: Bioinformatic Approach. International University Line, San Diego, CA.
Kolaskar, A. S. and Tongaonkar, P. C. (1990). A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett. 276, 172–174.
Benoiton, N. L., ed. (2006) Chemistry of Peptide Synthesis. Taylor & Francis, New York.
Howl, J., ed. (2005) Peptide Synthesis and Applications. Humana Press, Totowa, NJ.
Kates, S. A. and Albericio, F. (2000) Solid Phase Synthesis: A Practical Guide. Marcel Dekker, New York.
Fields, G. B. (1997) Solid-Phase Peptide Synthesis. Academic Press, New York.
Lateef, S., Gupta, S., Jayathilaka, G., Krishnanchettiar, S., and Lee, B. S. (2007) An improved protocol of coupling synthetic peptides to KLH for antibody production using MBS as bifunctional linker. J. Biomol. Tech. 18, 173–176.
Posnett, D., McGrath, H., and Tam, J. P. (1988). A novel method for producing anti-peptide antibodies. J. Biol. Chem. 263, 285–288.
Tam. J. P. (1988) Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system. PNAS USA 85, 5409–5413.
Westermeier, R. (2001) Electrophoresis in Practice. Wiley-VCH, Weinheim, Germany.
Hames, B. D. (1998) Electrophoresis of Protein. Oxford University Press, Oxford.
Acknowledgments
We thank the support of the Research Resources Center at the University of Illinois at Chicago.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Lee, BS., Huang, JS., Jayathilaka, G.D.L.P., Lateef, S.S., Gupta, S. (2010). Production of Antipeptide Antibodies. In: Schwartzbach, S., Osafune, T. (eds) Immunoelectron Microscopy. Methods in Molecular Biology, vol 657. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-783-9_7
Download citation
DOI: https://doi.org/10.1007/978-1-60761-783-9_7
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-782-2
Online ISBN: 978-1-60761-783-9
eBook Packages: Springer Protocols