The production and characterisation of monoclonal antibodies to myc, c-erbB-2 and EFG-receptor using a synthetic peptide approach.
Monoclonal antibodies to myc, c-erbB-2 and epidermal growth factor-receptor (EGF-R) were raised using a synthetic peptide approach. The antibodies were characterised by ELISA, immunoblotting, immunoprecipitation and immunocytochemical procedures against cognate peptide and native proteins. All of the monoclonal antibodies detected peptide-blockable bands of appropriate molecular weight (myc-p62/66 kDa, c-erbB-2-185kDa; EGF-R-150/170 kDa) on immunoblots. The monoclonal antibodies to c-erbB-2 and EGF-R immunostained subpopulations of tumour cells on sections of formalin-fixed, paraffin wax embedded human infiltrating and invasive ductal carcinomas of breast. Intense blood cell staining was observed with the EGF-R antibody. This staining was shown to be peptide blockable and may reveal a true localisation for the EGF-receptor protein, a closely-related (erbB) protein or a degradation product. The monoclonal antibody to a common peptide from the myc protein family was epitope scanned using a modification of the Geysen pin technique. Hexapeptide sequence Ala-Pro-Ser-Glu-Asp-Ile was found to be bound most strongly by the myc monoclonal antibody, and amino acids Pro2 and Glu4 were found to be essential for antibody binding. The use of synthetic peptides for the production of monoclonal antibodies with predetermined specificity, which may be precisely identified using the epitope scanning technique, is discussed.
Price
K M
KM
Division of Molecular Bioscience, Cambridge Research Biochemicals Ltd, Harston, UK.
Cuthbertson
A S
AS
Varndell
I M
IM
Sheppard
P W
PW
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000939:Epitopes / Q000032:analysis; D066246:ErbB Receptors / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D008969:Molecular Sequence Data; D016274:Oncogene Protein p55(v-myc); D018739:Oncogene Proteins v-erbB; D009856:Oncogene Proteins, Viral / Q000276:immunology*; D010455:Peptides / Q000276:immunology; D015682:Retroviridae Proteins, Oncogenic / Q000276:immunology*; D014407:Tumor Cells, Cultured; D014614:Vaccines, Synthetic
D000911:Antibodies, Monoclonal; D000939:Epitopes; D016274:Oncogene Protein p55(v-myc); D018739:Oncogene Proteins v-erbB; D009856:Oncogene Proteins, Viral; D010455:Peptides; D015682:Retroviridae Proteins, Oncogenic; D014614:Vaccines, Synthetic; D066246:ErbB Receptors