scholarly journals Purification of a Novel Anti-VEGFR2 Single Chain Antibody Fragmentand Evaluation of Binding Affinity by Surface Plasmon Resonance

2019 ◽  
Vol 9 (1) ◽  
pp. 64-69 ◽  
Author(s):  
Shirafkan Kordi ◽  
Mohammad Rahmati-Yamchi ◽  
Mehdi Asghari Vostakolaei ◽  
Abolfazl Barzegari ◽  
Jalal Abdolalizadeh
Keyword(s):  
Surface Plasmon Resonance ◽  
Phage Display ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Sodium Dodecyl ◽  
Scfv Antibody ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Phage Display Technology ◽  
Sds Page

Purpose: The single-chain variable fragment (scFv) domain of antibodies is now considered asone of the therapeutic tools that can be produced by phage display technology (PDT). Antibodypurification is one of the most important steps in antibodies production. The aim of study waspurification and characterization of anti-VEGFR2 scFv antibody fragments.Methods: After the coating of vascular endothelial growth factor receptor 2 (VEGFR2) peptidein ELISA microplates, the phage display library of Tomlinson was used for antibody isolation.The targeted scFv was purified by chromatography using a zeolite-based column. The purity andfunctional assessment of purified scFv were evaluated by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and western blotting techniques, respectively. Affinity bindingwas evaluated by surface plasmon resonance (SPR).Results: The desired scFv was selected after four stages of biopanning. SDS-PAGE analysisshowed a 28 kDa scFv with high purity (>90%). The western bloting analysis confirmed thebinding of produced scFv antibody to the desired peptide. The affinity binding of scFv antibodyanalyzed by SPR was about 60 μM.Conclusion: In this study, the novel scFv antibody against VEGFR2 peptide was purified bychromatography column containing zeolite. Based on our findings the produced antibody maybe applied for diagnosis or targeting of VEGFR2 in antibody-based therapy strategies.

Surface Plasmon Resonance‐Based Immunoassay for the Detection of Aflatoxin B1Using Single‐Chain Antibody Fragments

2005 ◽  
Vol 38 (3) ◽  
pp. 229-245 ◽  
Author(s):  
Lynsey Dunne ◽  
Stephen Daly ◽  
Andrew Baxter ◽  
Simon Haughey ◽  
Richard O'Kennedy
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Antibody Fragments ◽  
Single Chain ◽  
Single Chain Antibody

scholarly journals Selection of Linkers for a Catalytic Single-chain Antibody Using Phage Display Technology

1996 ◽  
Vol 271 (26) ◽  
pp. 15682-15686 ◽  
Author(s):  
Ying Tang ◽  
Ning Jiang ◽  
Cushrow Parakh ◽  
Donald Hilvert
Keyword(s):  
Phage Display ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Phage Display Technology ◽  
Display Technology ◽  
Selection Of

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

2019 ◽  
Author(s):  
Shirafkan Kordi ◽  
Mohammad Rahmati-Yamchi ◽  
Mehdi Asghari Vostakolaei ◽  
Ali Etemadie ◽  
Abolfazl Barzegari ◽  
...  
Keyword(s):  
Phage Display ◽  
Growth Factor Receptor ◽  
Phage Display Library ◽  
Vital Role ◽  
Scfv Antibody ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Phage Display Technology ◽  
Display Technology ◽  
Domain 3

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.

scholarly journals Automated Panning and Screening Procedure on Microplates for Antibody Generation from Phage Display Libraries

2009 ◽  
Vol 14 (3) ◽  
pp. 282-293 ◽  
Author(s):  
Laura Turunen ◽  
Kristiina Takkinen ◽  
Hans Söderlund ◽  
Timo Pulli
Keyword(s):  
Phage Display ◽  
High Efficiency ◽  
Magnetic Bead ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Phage Display Technology ◽  
Antibody Phage ◽  
Antibody Phage Display ◽  
Display Technology

Antibody phage display technology is well established and widely used for selecting specific antibodies against desired targets. Using conventional manual methods, it is laborious to perform multiple selections with different antigens simultaneously. Furthermore, manual screening of the positive clones requires much effort. The authors describe optimized and automated procedures of these processes using a magnetic bead processor for the selection and a robotic station for the screening step. Both steps are performed in a 96-well microplate format. In addition, adopting the antibody phage display technology to automated platform polyethylene glycol precipitation of the enriched phage pool was unnecessary. For screening, an enzyme-linked immunosorbent assay protocol suitable for a robotic station was developed. This system was set up using human γ-globulin as a model antigen to select antibodies from a VTT naive human single-chain antibody (scFv) library. In total, 161 γ-globulin-selected clones were screened, and according to fingerprinting analysis, 9 of the 13 analyzed clones were different. The system was further tested using testosterone bovine serum albumin (BSA) and β-estradiol-BSA as antigens with the same library. In total, 1536 clones were screened from 4 rounds of selection with both antigens, and 29 different testosterone-BSA and 23 β-estradiol-BSA binding clones were found and verified by sequencing. This automated antibody phage display procedure increases the throughput of generating wide panels of target-binding antibody candidates and allows the selection and screening of antibodies against several different targets in parallel with high efficiency. ( Journal of Biomolecular Screening 2009:282-293)

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