title: Production and Characterization of Novel Camel Single Domain Antibody Targeting Mouse Vascular Endothelial Growth Factor.
abstract: Camel single domain antibody known as Nanobody™ refers to a novel class of monoclonal antibodies with appropriate pharmacological properties. Nanobody is an antigen-binding site of camel heavy chain antibody also known as VHH. Expression in a microbial system, stability in difficult conditions and extremes of PH, and nanomolar affinity to target an appropriate drug format makes Nanobody a potential for drug discovery. Needs for Nanobody function evaluation in animal models turned our interest to develop anti-mouse vascular endothelial growth factor (mVEGF) Nanobodies using phage display as a potent technique in the isolation of antibodies. Isolation of anti-mVEGF Nanobodies was performed on Camelus dromedarius immune library through four consecutive rounds of biopanning on immobilized mVEGF. Enrichment of the Nanobody library was monitored by polyclonal phage-ELISA, and specific Nanobodies were selected using periplasmic extract-ELISA. Selected Nanobodies were expressed in WK6 Escherichia coli cells and purified using immobilized metal affinity chromatography. Specificity and affinity of selected Nanobodies were evaluated on immobilized mVEGF. Results demonstrated the successful enrichment of the Nanobody library. Two clones named Nb5 and Nb10 were selected through screening procedures according to their signal value in periplasmic extract-ELISA. Selected Nanobodies specifically reacted to mVEGF, but cross-reactivity with other antigens was not observed. Evaluated affinity for the Nanobodies was in nanomolar range. Taken together, according to the results, the selected Nanobodies promise to be a novel tool in research and for further development of diagnostic or therapeutic purposes in pharmaceutical science.
authors.affiliation: 1 Biotechnology Research Center, Venom & Biotherapeutics Molecules Laboratory, Pasteur Institute of Iran , Tehran, Iran .
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Kazemi-Lomedasht
authors.affiliation: 1 Biotechnology Research Center, Venom & Biotherapeutics Molecules Laboratory, Pasteur Institute of Iran , Tehran, Iran .
authors.forename: Mahdi
authors.initials: M
authors.lastname: Behdani
authors.affiliation: 2 National Cell Bank of Iran, Pasteur Institute of Iran , Tehran, Iran .
authors.forename: Mahdi
authors.initials: M
authors.lastname: Habibi-Anbouhi
authors.affiliation: 1 Biotechnology Research Center, Venom & Biotherapeutics Molecules Laboratory, Pasteur Institute of Iran , Tehran, Iran .
authors.forename: Delavar
authors.initials: D
authors.lastname: Shahbazzadeh
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D002162:Camelus / Q000276:immunology*; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D019151:Peptide Library; D061905:Single-Domain Antibodies / Q000096:biosynthesis / Q000276:immunology*; D042461:Vascular Endothelial Growth Factor A / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D019151:Peptide Library; D061905:Single-Domain Antibodies; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse