Epitope mapping by region-specified PCR-mutagenesis

Methods Mol Biol. 2009:524:305-13. doi: 10.1007/978-1-59745-450-6_22.

Abstract

We will describe a procedure to map epitopes on a protein against monoclonal IgGs. In this procedure, we amplified and mutagenized the entire or a part of the protein. Then, a DNA region encoding the protein was cut out by a restriction enzyme and ligated into a lambda-gt11 expression vector to construct a library. Thus, the protein is expressed as a fusion protein with beta-galactosidase. Protein in plaques obtained by phages derived from the library were tested for cross-reactivities by means of immunoblotting experiments.

MeSH terms

  • Antibodies, Monoclonal / immunology
  • Antigens / genetics
  • Antigens / immunology
  • Base Sequence
  • Cloning, Molecular
  • Cross Reactions
  • DNA Primers / genetics
  • Epitope Mapping / methods*
  • Gene Library
  • Humans
  • Immunoglobulin G / immunology
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Point Mutation
  • Polymerase Chain Reaction / methods*
  • Rec A Recombinases / genetics
  • Rec A Recombinases / immunology

Substances

  • Antibodies, Monoclonal
  • Antigens
  • DNA Primers
  • Immunoglobulin G
  • Rec A Recombinases