Epitope mapping by region-specified PCR-mutagenesis.
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.
Mikawa
Tsutomu
T
Biometal Science Laboratory, RIKEN SPring-8 Center, Hyogo, Japan.
Lkeda
Masayuki
M
Shibata
Takehiko
T
D000911:Antibodies, Monoclonal / Q000276:immunology; D000941:Antigens / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D003429:Cross Reactions; D017931:DNA Primers / Q000235:genetics; D018604:Epitope Mapping / Q000379:methods*; D015723:Gene Library; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011938:Rec A Recombinases / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000941:Antigens; D017931:DNA Primers; D007074:Immunoglobulin G; D011938:Rec A Recombinases