Selection of Single-Chain Variable Fragment Antibodies to Black Currant Reversion Associated Virus from a Synthetic 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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Single chain variable fragment antibodies against Shiga toxins isolated from a human antibody phage display library

Vaccine ◽

10.1016/j.vaccine.2011.05.093 ◽

2011 ◽

Vol 29 (33) ◽

pp. 5340-5346 ◽

Cited By ~ 14

Author(s):

Paola Neri ◽

Naoko Shigemori ◽

Susumu Hamada-Tsutsumi ◽

Kentaro Tsukamoto ◽

Hideyuki Arimitsu ◽

...

Keyword(s):

Phage Display ◽

Phage Display Library ◽

Human Antibody ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Shiga Toxins ◽

Antibody Phage ◽

Antibody Phage Display

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Isolation of Lawsonia intracellularis specific single-chain Fv antibody fragments from phage display library

Research in Veterinary Science ◽

10.1016/j.rvsc.2006.11.002 ◽

2007 ◽

Vol 83 (1) ◽

pp. 85-90 ◽

Cited By ~ 1

Author(s):

K. Dezorzová-Tomanová ◽

D. Molinková ◽

M. Pekarová ◽

V. Celer ◽

J. Smola

Keyword(s):

Phage Display ◽

Phage Display Library ◽

Antibody Fragments ◽

Lawsonia Intracellularis ◽

Single Chain ◽

Single Chain Fv ◽

Fv Antibody ◽

Single Chain Fv Antibody

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Screening Antibody Libraries with Colony Assay Using scFv-Alkaline Phosphatase Fusion Proteins

Molecules ◽

10.3390/molecules25122905 ◽

2020 ◽

Vol 25 (12) ◽

pp. 2905

Author(s):

Yoshiro Hanyu ◽

Mieko Kato

Keyword(s):

Alkaline Phosphatase ◽

Fusion Proteins ◽

Direct Detection ◽

Screening Methods ◽

Single Chain Variable Fragment ◽

Antibody Library ◽

Single Chain ◽

Library Size ◽

Colony Assay ◽

Antibody Libraries

Screening antibody libraries is an important step in establishing recombinant monoclonal antibodies. The colony assay can identify positive clones without almost any false-positives; however, its antibody library is smaller than those used in other recombinant screening methods such as phage display. Thus, to improve the efficiency of colony assays, it is necessary to increase library size per screening. Here, we report developing a colony assay with single-chain variable fragment (scFv) fused to the N-terminus of bacterial alkaline phosphatase (scFv-PhoA). The scFv-PhoA library was constructed in an expression vector specifically designed for this study. Use of this library allowed the successful and direct detection of positive clones exhibiting PhoA activity, without the need for a secondary antibody. Colony assay screening with scFv-PhoA is simple, rapid, offers a higher success rate than previous methods based on scFv libraries, and—most importantly—it enables high-throughput procedures.

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Specific detection of myeloma plasma cells using anti-idiotypic single chain antibody fragments selected from a phage display library

Leukemia ◽

10.1038/sj.leu.2401123 ◽

1998 ◽

Vol 12 (8) ◽

pp. 1295-1302 ◽

Cited By ~ 5

Author(s):

PMW Willems ◽

RMA Hoet ◽

ELPG Huys ◽

JMH Raats ◽

EJBM Mensink ◽

...

Keyword(s):

Phage Display ◽

Plasma Cells ◽

Phage Display Library ◽

Antibody Fragments ◽

Single Chain ◽

Specific Detection ◽

Single Chain Antibody

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

Science China Life Sciences ◽

10.1007/s11427-016-5077-7 ◽

2016 ◽

Vol 59 (7) ◽

pp. 686-693 ◽

Cited By ~ 2

Author(s):

Qian Wu ◽

Xiaodong Wang ◽

Yong Gu ◽

Xiao Zhang ◽

Yao Qin ◽

...

Keyword(s):

Type 1 Diabetes ◽

Phage Display ◽

Phage Display Library ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Screening And Identification

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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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