Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library

Single-chain variable fragment (scFv) antibodies that bind to black currant reversion associated virus (BRAV) were obtained from a synthetic phage display antibody gene library without recourse to animal immunizations. Several different BRAV-specific phage scFv were obtained quickly, after only three rounds of selection against immobilized virus antigen. The phage scFv gave enzyme-linked immunosorbent assay (ELISA) absorbance values that were greater than seven times the control healthy plant extracts. In contrast, comparative tests using a rabbit antiserum failed, because unacceptably high background values were obtained with healthy plant extracts. Two of the scFv were subcloned into the pDAP2 vector for the rapid and efficient production of scFv-alkaline phosphatase fusion proteins. Functional fusion proteins were obtained after expression in Escherichia coli, and preparations from periplasmic extracts detected BRAV in ELISA. The results demonstrate that antibody fragments obtained from a synthetic phage display library are useful research tools, and they proved to be a viable practical alternative when traditional antisera failed to detect BRAV, a weak immunogen. Furthermore, the genetic fusion of antibody fragments to alkaline phosphatase obviates the need for further chemical coupling procedures, and the fusion proteins can be obtained cheaply.

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

Iranian Journal of Allergy Asthma and Immunology ◽

10.18502/ijaai.v18i3.1122 ◽

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.

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Expression and Characterization of Recombinant Interleukin-21 Receptor and Its Targeting Single-Chain Variable Fragment Antibodies Selected from a Human Phage Display Library

DNA and Cell Biology ◽

10.1089/dna.2012.1728 ◽

2012 ◽

Vol 31 (10) ◽

pp. 1541-1548 ◽

Cited By ~ 1

Author(s):

Qinhang Wu ◽

Juan Zhang ◽

Chen Luo ◽

Tao Zhang ◽

Tong Wang ◽

...

Keyword(s):

Phage Display ◽

Phage Display Library ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Interleukin 21

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Selection of single chain variable fragment (scFv) antibodies from a hyperimmunized phage display library for the detection of the antibiotic monensin

Journal of Immunological Methods ◽

10.1016/j.jim.2010.06.015 ◽

2010 ◽

Vol 360 (1-2) ◽

pp. 103-118 ◽

Cited By ~ 16

Author(s):

Shokouh Makvandi-Nejad ◽

Claudia Sheedy ◽

Linda Veldhuis ◽

Gabrielle Richard ◽

J. Christopher Hall

Keyword(s):

Phage Display ◽

Phage Display Library ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Selection Of

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Characterization of LL25X, a Newly Isolated Anti-SIV Monoclonal Antibody, to Determine Binding Specificity

Elements ◽

10.6017/eurj.v13i1.9604 ◽

2017 ◽

Vol 13 (1) ◽

Author(s):

Zackary Tajin Park

Keyword(s):

Phage Display ◽

Mammalian Cells ◽

Expression Vector ◽

Restriction Enzymes ◽

Phage Display Library ◽

Single Chain ◽

Phage Display Technology ◽

Mammalian Expression ◽

Mammalian Expression Vector ◽

Single Chain Fv

A phage display library was previously constructed from an SIV-infected rhesus macaque. Several single chain Fv (scFv), including SU24, SU343 and LL25X, were selected using phage display technology. Sequences corresponding to SU24, SU343 and LL25X were optimized for expression in a mammalian system and commercially synthesized. SU24 and SU343 had previously been cloned into a mammalian expression vector. In this study, we aimed to characterize the specificity of SU24, SU343, and LL25X.. The codon-optimized version of the scFv LL25X gene sequence was cloned into a mammalian expression vector (pCEP4). LL25X DNA was amplified by PCR, and the PCR product and mammalian expression vector were both digested with KpnI/SapI restriction enzymes. Digested fragments were purified, and the fragments were ligated using T4DNA ligase. E. coli cells were transformed with the ligation reaction. Single colonies were selected on LB agar plates containing the selective antibiotic (ampicillin). Positive colonies were identified after DNA mini-preparation and test-digestion with KpnI and SapI. Sanger sequencing confirmed cloning results and DNA sequence accuracy. Following transfection of mammalian cells (293T), LL25X-Fc cells, and purifying our protein, the binding of LL25X-Fc to the SIV gp140 envelope protein was confirmed via ELISA and Western Blotting.

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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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