Preparation of single chain variable fragment of MG7mAb by phage display technology

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.

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Production of a Recombinant Anti-Human CD4 Single-Chain Variable-Fragment Antibody Using Phage Display Technology and Its Expression in Escherichia coli

Journal of Microbiology and Biotechnology ◽

10.4014/jmb.1010.10022 ◽

2011 ◽

Vol 21 (5) ◽

pp. 529-535 ◽

Cited By ~ 8

Author(s):

Arash Babaei

Keyword(s):

Escherichia Coli ◽

Phage Display ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Phage Display Technology ◽

Expression In Escherichia Coli ◽

Display Technology

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Characterization of Monoclonal Antibodies against Bovine herpesvirus type 1 selected by Phage Display Technology

Semina Ciências Agrárias ◽

10.5433/1679-0359.2017v38n6p3915 ◽

2017 ◽

Vol 38 (6) ◽

pp. 3915

Author(s):

Greice Japolla ◽

Ana Flávia Batista Penido ◽

Greyciele Rodrigues Almeida ◽

Luiz Artur Mendes Bataus ◽

Jair Pereira Cunha Junior ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Phage Display ◽

Bovine Herpesvirus ◽

Single Chain ◽

Phage Display Technology ◽

Herpesvirus Type ◽

Wide Range ◽

Display Technology ◽

Bovine Herpesvirus Type 1

The specificity of monoclonal antibodies (mAbs) to desired targets makes these molecules suitable for therapeutic and diagnostic uses against a wide range of pathogens. Phage display antibody libraries offer one method by which mAbs can be selected for, without the use of conventional hybridoma technology. In this work, phage display technology was used to construct, select and characterize a combinatorial single chain fragment variable (scFv) antibody library against bovine herpesvirus type 1 (BoHV-1) from the immune repertoire of chickens immunized with the virus. In silico analysis of the hypervariable domains of the antibody heavy chains revealed a high frequency of scFv fragments with low variability, suggesting that selection had probably been carried out and favored by a few im-munogenic viral antigens. The reactivity of the scFv fragments selected against BoHV-1 was demon-strated by Phage-ELISA. A significant increase in antibody reactivity to the target was observed after six rounds of library selection, showing its potential use as a molecule for BoHV-1 diagnosis. The strategy described here opens up a field for the use of phage display as a tool for selection of mono-clonal antibodies that could be used for theranostic applications against infectious and parasitic dis-eases of veterinary interest.

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Strategies to improve scFvs as crystallization chaperones suggested by analysis of a complex with the human PHD-bromodomain SP140

10.1101/767376 ◽

2019 ◽

Author(s):

Michael Fairhead ◽

Charlotta Preger ◽

Edvard Wigren ◽

Claire Strain-Damerell ◽

Elena Ossipova ◽

...

Keyword(s):

Structural Biology ◽

Crystal Packing ◽

Specific Protein ◽

Antibody Fragments ◽

Complex Structures ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Phage Display Technology ◽

Disease States ◽

Display Technology

AbstractAntibody fragments have great potential as crystallization chaperones for structural biology due to their ability to either stabilise targets, trap certain conformations and/or promote crystal packing. Here we present an example of using a single-chain variable fragment (scFv) to determine the previously unsolved structure of the multidomain protein SP140. This nuclear leukocyte-specific protein contains domains related to chromatin-mediated gene expression and has been implicated in various disease states. The structure of two of the domains (PHD-bromodomain) was solved by crystallizing them as a complex with a scFv generated by phage display technology. SP140 maintains a similar overall fold to previous PHD-bromodomains and the scFv CDR loops predominately interact with the PHD, while the framework regions of the scFv makes numerous interactions with the bromodomain. Analysis of our and other complex structures suggest various protein engineering strategies that might be employed to improve the usefulness of scFvs as crystallization chaperones.

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