Production and Characterization of Monoclonal Antibodies Against the Dimerization Domain of Human HER۲

Background: Human Epidermal Growth factor Receptor ۲ (HER۲), also known as ErbB۲ is a ۱۸۵ kDa protein belonging to the Human Epidermal Receptor (HER) family of tyrosine kinase receptors overexpressed in ۲۰% - ۳۰% of patients with breast cancer. Similar to other members of the HER family, HER۲ glycoprotein comprises of multiple domains including an extracellular ligand-binding domain, a single transmembrane domain and a cytoplasmic domain with tyrosine kinase activity. The extracellular domain of HER۲ with ۶۳۲ amino acids is composed of four subdomains (I - IV); subdomains I and III form a ligand binding site, and cysteine-rich subdomains II and IV play an important role in dimerization of the receptor. Materials and Methods: Primarily, BALB/c mice were immunized with a ۳۰-aminoacid peptide as a part of the human HER۲ subdomain II. Splenocytes from hyperimmunized mice were fused with myeloma cells (SP۲/۰), selected in hypoxanthine-aminopterin-thymidine (HAT) medium, and screened by indirect Enzyme-Linked Immunosorbent Assay (ELISA). Secreted MAbs were characterized according to isotypes, reactions with the native HER۲ in SKBR۳ cells by western blotting, and in tissue sections from HER۲ positive breast cancer specimens by Immunohistochemistry (IHC). Conclusions: The results of both western blotting and Immunohistochemistry showed that native HER۲ can be detected with ۱F۱ monoclonal antibody.