Epitope Mapping with Gene Fragment Libraries

Part of the Springer Lab Manuals book series (SLM)

Abstract

Linear epitopes, recognized by a large number of antibodies, are easily identified by expression of small fragments of the known antigen at the tip of filamentous phage. Immobilized antibodies, incubated with such a library of peptide-displaying phage, bind selectively to the fragments containing the epitope, therefore immobilizing the peptide-phage construct as well. Unspecific non-binding phage can then be washed away. Finally, by eluting the epitope-displaying phage following their propagation in E. coli, repetitive rounds of panning are performed to further enrich for specific phage. After approximately three rounds of panning, several single phage clones are amplified and the antigen-encoding inserts are then sequenced. Aligning the insert sequences with the antigen sequence identifies the region in which the epitope lies. The comparison of different fragments usually yields overlapping sequences, each containing the epitope, hence narrowing down the number of amin0 acids essential for binding. Compared to the previously described random peptide libraries, the gene-fragment phage display offers the major advantage of using the correct antigen of an antibody to find its epitope, thus eliminating guesses and sequence comparisons with unknown or irrelevant proteins. Therefore, the chances of finding one or more sequences binding to the antibody that are unrelated to the antigen (mimotopes) are drastically reduced in favor of the correct epitope.

Keywords

Vortex Glycerol Corn Agar Electrophoresis 

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Copyright information

© Springer-Verlag Berlin Heidelberg 2001

Authors and Affiliations

  1. 1.School of Medicine, Dept. of Molecular Biophysics and BiochemistryYale UniversityNew HavenUSA

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