Submitted: 12 May 2015
Accepted: 31 May 2015
First published online: 20 Jul 2015
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Avicenna J Med Biochem. 2015;3(2): e29918.
doi: 10.17795/ajmb-29918
  Abstract View: 358
  PDF Download: 343
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Research Article

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

Mohammad Nadri 1, Mohammad Soukhtanloo 1,2, Amir Hosein Jafarian 2, Mahboobeh Alamdari 1, Mohsen Sisakhti 1, Tayebeh Kianoush 1, Farnaz Zahedi Avval 1,3 *

1 Department of Biochemistry, School of Medicine, Mashhad University of Medical Sciences, Mashhad, IR Iran
2 Cancer Molecular Pathology Research Center, Ghaem Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, IR Iran
3 Biochemistry & Nutrition Research Center, Mashhad University of Medical Sciences, Mashhad, IR Iran
*Corresponding author: Farnaz Zahedi Avval, Department of Biochemistry, School of Medicine, Mashhad University of Medical Sciences, P. O. Box: 917794-8564, Mashhad, IR Iran. Tel: +98-5138002365, Fax: +98-5138828574, Email: zahediaf@mums.ac.ir

Article

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.

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