Báo cáo khoa học: Identification of the epitope of a monoclonal antibody that disrupts binding of human transferrin to the human transferrin receptor

Identification of the epitope of a monoclonal antibody that disrupts binding of human transferrin to the human transferrin receptor

Authors: Evelyn M. Teh, Jeff Hewitt, Karen C. Ung, Tanya A. M. Griffiths, Vinh Nguyen, Sara K. Briggs, Anne B. Mason, and Ross T. A. MacGillivray

Field: Biochemistry and Molecular Biology

Document Content: This study focuses on identifying the specific epitope of a monoclonal antibody (mAb) F11 that inhibits the binding of human transferrin (hTF) to the human transferrin receptor (TFR). By constructing and expressing various regions of hTF as glutathione S-transferase (GST) fusion proteins in Escherichia coli, and subsequently analyzing them with mAb F11 via immunoblotting, the researchers localized the F11 epitope to the C1 domain of hTF, specifically within residues 365-401. Further analysis, including mutagenesis studies mimicking pig and mouse transferrin sequences, confirmed this localization. These findings suggest that this region of hTF plays a crucial role in its interaction with the TFR.

Detailed Table of Contents:

• Introduction
• Results
• TFR binding studies
• Analysis of IPTG-induced fusion protein expression
• Epitope mapping for mAb F11
• Studies with synthetic peptide
• Crossreactivity of the mAb F11
• Discussion
• Experimental procedures
• Materials
• TFR binding studies
• Cloning of hTF fragments
• Cloning of hTF fragments into the pGEX 4T3 vector
• IPTG induction of GST-hTF fusion proteins
• Gel electrophoresis and western blotting
• Peptide construction and immunoassay
• Molecular mapping
• Acknowledgements
• References