Isolation of a novel Anti-KDR3 Single-chain Variable Fragment Antibody from a Phage Display Library

Shirafkan   Kordi; Mohammad  Rahmati-Yamchi; Mehdi  Asghari Vostakolaei; Ali  Etemadie; Abolfazl   Barzegari; Jalal   Abdolalizadeh

doi:10.18502/ijaai.v18i3.1122

Shirafkan Kordi
Mohammad Rahmati-Yamchi
Mehdi Asghari Vostakolaei
Ali Etemadie
Abolfazl Barzegari
Jalal Abdolalizadeh

DOI: https://doi.org/10.18502/ijaai.v18i3.1122

Keywords: Monoclonal antibody; Kinase insert domain receptor (KDR) 3; Phage display; Single-chain variable fragment (scFv); Vascular endothelial growth factor receptor-2 (VEGFR2)

Abstract

Vascular endothelial growth factor receptor 2 (VEGFR-2) is known as one of the important antigens playing a vital role in angiogenesis. In this study, phage display technology (PDT) was used to produce a single-chain variable fragment (scFv) antibody against a region of the domain 3 in VEGFR-2 called kinase insert domain receptor 3 (KDR3).

After designing the KDR3 peptide and biopanning, a colony was chosen for scFv antibody expression. Following expression and purification; western blotting, dot blotting and immunofluorescence (IF) were used to evaluate the antibody function. Surface plasmon resonance (SPR) was also employed to measure affinity of produced antibody.

Once a colony was selected and transferred to the expression host, the scFv antibody was expressed in the expected range of 28 kDa. Using a designed chromatography column, antibody purification was found to be about 95%.

In this study, a novel scFv with the capability of binding to KDR3 was isolated and purified and its intracellular function was investigated and verified.