Characterization of two monoclonal antibodies against the COOH-terminal part of the human epidermal growth factor receptor and potential clinical use

Two monoclonal antibodies of the IgG2 subclass, designated A 01 and B 11, were prepared against two synthetic peptides corresponding to the COOH-terminal sequence of the human epidermal growth factor receptor (EGF-R), in order to detect EGF-R in a radioimmunometric assay and by immunohistochemistry. Characterization of these Mabs showed that they recognized two different epitopes on the original peptides with K d of 1.7 × 10 −8 M and 1.3 × 10 −7 M, respectively, without crossreaction. The A 431 antigen recognized by A 01 and B 11 had an apparent molecular weight of approximatively 170,000 and was able to specifically link to EGF. Thus, A 01 and B 11 are directed against an antigenic site on the human EGF-R. With Western blot analysis and immunostaining, A 01 was shown to be EGF-R specific. In addition to the EGF-R, B 11 recognized two unidentified soluble proteins present in the cytoplasm of the SKBR-3 cell line but different from the c- erb B-2 oncoprotein expressed by these cells. Mabs A 01 and B 11 were used in an IRMA for the determination of EGF-R using the A 431 cell line as a source of EGF-R. Mab A 01 was also shown to be a useful tool for immunohitochemical detection of EGF-R.