Summary
Multiple antigenic peptides (MAPs) can be efficiently separated on sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to a nitrocellulose membrane for immunoblotting. MAPs involve a hepta lysine core with end groups for anchoring multiple copies of the same synthetic peptide. MAPs are amenable to staining with Coomassie and silver on SDS polyacrylamide gels as well as by Fast Green on a blotted nitrocellulose membrane. They lend themselves to analysis on an immunoblot as they behave like low molecular weight proteins. Affinity immunoblotting for analysis of antibody clonotype distribution has also been carried out using these peptides.
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References
Tam, J.P. (1988) Synthetic peptide vaccine design: synthesis and properties of high-density multiple antigenic peptide system. Proc Natl Acad Sci USA 85, 5409–5413.
Posnett, D.N., McGrath H., and Tam J.P. (1988) A novel method for producing anti-peptide antibodies. Production of site specific antibodies to the cell antigen receptor beta- chain. J Biol Chem. 263, 1719–1725.
Troalen, F., Razafindratsita A., Puisieux A., Voeltzel T., Bohuon C., Bellet D., (1990) Structural probing of human lutropin using antibodies raised against synthetic peptides constructed by classical and multiple antigen peptide system approaches. Mol Immunol. 27, 363–368.
Munesinghe, D.Y., Clavijo P., Calle M.C., Nuzzenzweig R.S., and Nardin E. (1991) Immunogenicity of multiple antigen peptides (MAP) containing T and B cell epitopes of the repeat region of the P. falciparum circumsporazoite protein. Eur J Immunol. 21, 3015–3020.
McLean, G.W., Owsianka A.M., Subak-Sharpe J.H., and Marsden H.S. (1991) Generation of anti-peptide and anti-protein sera. Effect of peptide presentation on immunogenicity. J Immunol Meth. 137, 149–157.
Habluetzel, A., Pessi A., Bianchi E., Rotigliano G., and Esposito F. (1991) Multiple antigen peptides for specific detection of antibodies to a malaria antigen in human sera. Immunol Lett. 30, 75–80.
Francis, M.J., Hastings G.Z., Brown F., McDermed J., Lu Y.-A., and Tam J.P. (1991) Immunological evaluation of the multiple antigen peptide (MAP) system using the major immunogenic site of foot-and-mouth disease virus. Immunology 73, 249–254.
James, J.A., Gross T., Scofield R.H., and Harley J.B. (1995) Immunoglobulin epitope spreading and autoimmune disease after peptide immunization: Sm B/B’-derived PPPGMRPP and PPGIRPP induce spliceosome autoimmunity. J Exp Med. 181, 453–461.
Tam, J.P. and Zavala F. (1989) A novel approach to increase detection sensitivity of synthetic peptides in solid-phase immunoassays. J Immunol Meth. 124, 53–61.
Briand, J.-P., Barin C., Regenmortel M.H.V.V., and Muller S. (1992) Application and limitations of the multiple antigenic peptide (MAP) system in the production and evaluation of anti-peptide and anti-protein antibodies. J Immunol Meth. 156, 255–265.
Tam, J.P. and Lu Y.-A. (1989) Vaccine engineering: Enhancement of immunogenicity of synthetic peptide vaccines related to hepatitis in chemically defined models consisting of T- and B- cell epitopes. Proc Natl Acad Sci USA 86, 9084–9088.
Scofield R.H., Kurien B.T., Zhang F., Mehta P., Kaufman K., Gross T., . (1999) Protein-protein interaction of the Ro- ribonucleoprotein particle using multiple antigenic peptides. Mol Immunol. 36, 1093–1106.
Scofield R.H., Kurien B.T., Gross J.K., Reed N.B., Taylor A.K., Dominguez N., . (2004) Interaction of calcium and Ro60: increase of antigenicity. Mol Immunol. 41, 809–816.
Scofield, R.H., Kurien B.T., and Reichlin M. (1997) Immunologically restricted and inhibitory anti-Ro/SSA in monozygotic twins. Lupus 6, 395–398.
McLean G.W., Cross A.M., Munns M.S., and Marsden H.S. (1992) Rapid attachment of a helper T cell epitope to branched peptides by fragment condensation to give enhanced immunogenicity. J Immunol Methods 155, 113–120.
Yao, Z.J., Kao M.C.C., Loh K.C., and Chung M.C.M. (1994) Antibody against branched epitope as an affinity ligand to separate the parent protein. J Chromatogr A. 679, 190–194.
Deutscher, S.L., Harley J.B., and Keene J.D. (1988) Molecular analysis of the 60 kD human Ro ribonucleoprotein. Proc Natl Acad Sci USA 85, 9479–9483.
Ben-Chetrit, E., Gandy B.J., Tan E.M., and Sullivan K.F. (1989) Isolation and characterization of a cDNA clone encoding the 60 kDa component of the human SS- A/Ro ribonucleoprotein autoantigen. J Clin Invest. 83, 1284–1292.
James, J.A., Gross T., Scofield R.H., and Harley J.B. (1995) Immunoglobulin epitope spreading and autoimmune disease after peptide immunization: Sm B/B’-derived PPPGMRPP and PPGIRPP induce spliceosome autoimmunity. J Exp Med. 181, 453–461.
Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680–685.
Towbin, H., T. and Gordon J.. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and applications. Proc Natl Acad Sci USA 76, 4350–4354.
Kurien B.T., Jackson K., and Scofield R.H. (1998) Immunoblotting of multiple antigenic peptides. Electrophoresis 19, 1659–1661.
Wu M., Stockley P.G., and Martin W.J. 2nd. (2002) An improved Western blotting technique effectively reduces background. Electrophoresis 23, 2373–2376.
Acknowledgement
This work was supported by NIH grant ARO1844 and Oklahoma Center for the Advancement of Science and Technology to Dr. Hal Scofield, OMRF, OKC, OK, USA.
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Kurien, B.T. (2009). Strip Immunoblotting of Multiple Antigenic Peptides to Nitrocellulose Membrane. In: Kurien, B., Scofield, R. (eds) Protein Blotting and Detection. Methods in Molecular Biology, vol 536. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-542-8_21
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DOI: https://doi.org/10.1007/978-1-59745-542-8_21
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