METHODS FOR GENERATING PHOSPHORYLATION SITE-SPECIFIC IMMUNOLOGICAL REAGENTS.

摘要

The present invention provides methods for generating phosphorylation site-specific immunological reagents. More specifically, a phosphopeptide mimetic is incorporated into a polypeptide in place of a phosphorylated amino acid. The polypeptide is used as antigen by standard methods to generate either monoclonal or polyclonal antibodies which cross-react with naturally phosphorylated polypeptide. The phosphopeptide mimetic preferably contains a non-hydrolyzable linkage from the appropriate carbon atom of the amino acid to a phosphate group. A preferred linkage is a CF2 group. Such a linkage is used to generate the phosphoserine mimetic F2Pab, which is incorporated into a polypeptide sequence derived from p53 to produce antibodies which recognize a specific phosphorylation state of p53. A CF2 group linkage is also used to produce antibodies to produce the phosphothreonine mimetic F2Pmb, and to produce the phosphotyrosine mimetic, F2Pmp.