Epitope mapping of SHP-1 monoclonal antibodies using peptide phage display.
We have characterized the binding epitopes of four monoclonal antibodies for SHP-1, an SH2 domain containing protein tyrosine phosphatase, using two phage displayed random peptide libraries. Three of the antibodies are directed against the phosphatase domain of the molecule and the fourth is toward the NH2-terminal part of the second SH2 domain. The first two antibodies recognize the sequence NANY, amino acid 305 to amino acid 308, numbered in the non haematopoietic form of human SHP-1 sequence. The third antibody binds the sequence P Y W P (amino acids 365 to 368) located toward the middle of the phosphatase domain of the enzyme. The fourth antibody is directed against the first two amino acids, W Y (amino acids 112 and 113), of the second SH2 domain. The specificities of these antibodies are demonstrated by ELISA and western blot using different protein constructs expressed in bacteria. All the antibodies can detect wild type SHP-1, expressed in 293 cells, by western blot analysis, both under denaturing conditions as well as following renaturation. The data presented here show that the antibodies characterized in this study are raised against linear epitopes and suggest that these epitopes are accessible from the outside in the native SHP-1 molecule.
Murthy
K K
KK
Pharmaceutical Biotechnology Sector, Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada. kishore.murthy@nrc.ca
Shen
S H
SH
Banville
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D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001435:Bacteriophages; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D019151:Peptide Library; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D018909:src Homology Domains
D000911:Antibodies, Monoclonal; D047908:Intracellular Signaling Peptides and Proteins; D019151:Peptide Library; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases