Production and Characterization of Monoclonal Antibodies Against the Dimerization Domain of Human HER2

Abstract

Background: Human Epidermal Growth factor Receptor 2 (HER2), also known as ErbB2 is a 185 kDa protein belonging to the Human Epidermal Receptor (HER) family of tyrosine kinase receptors overexpressed in 20% -30% of patients with breast cancer. Similar to other members of the HER family, HER2 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 HER2 with 632 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. Objectives: In this study we aimed to produce murine Monoclonal Antibodies (MAbs) with the ability of specific recognition of the HER2 dimerization arm. Materials and Methods: Primarily, BALB/c mice were immunized with a 30-aminoacid peptide as a part of the human HER2 subdomain II. Splenocytes from hyperimmunized mice were fused with myeloma cells (SP2/0), 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 HER2 in SKBR3 cells by western blotting, and in tissue sections from HER2 positive breast cancer specimens by Immunohistochemistry (IHC). Results: Isotype of 1F1 clone was determined to be IgG1, which reacted with native protein in the western blot experiment and stained 20% of the membrane of neoplastic cells overexpressing HER2 with 3+ grade. However, 3L5 clone showed a low reaction (10%) with native HER2 in immunohistochemistry. Conclusions:The results of both western blotting and Immunohistochemistry showed that native HER2 can be detected with 1F1 monoclonal antibody.

A One-Step Plasma Assisted Synthesis of Gold Nanoparticles and Simultaneous Linker-Free Conjugation with Nestin: An In Vitro Study of Cellular toxicity

“…To prepare our experiment samples to investigate the linker-free conjugation, we proceeded as follows. Synthetic Nestin (NES) 8MAPs peptide was ordered from PADZA company with 100 μg/ml concentrations (in PBS solution) [52], which subsequently will be mentioned as the primary NES solution to conjugate it on our nanoparticles. NES molecular weight (∼16 kDa), schematic structure, and sequence are shown in Figures 1C, E, and F, respectively.…”