Protein III-based single-chain antibody phage display using bacterial cells bearing an additional genome of a gene-III-lacking helper phage

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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Construction of Recombinant Single Chain Variable Fragment (ScFv) Antibody Against Superantigen for Immunodetection Using Antibody Phage Display Technology

Superantigens - Methods in Molecular Biology ◽

10.1007/978-1-4939-3344-0_17 ◽

2015 ◽

pp. 207-225 ◽

Cited By ~ 2

Author(s):

Pawan Kumar Singh ◽

Ranu Agrawal ◽

D. V. Kamboj ◽

Lokendra Singh

Keyword(s):

Phage Display ◽

Scfv Antibody ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Phage Display Technology ◽

Antibody Phage ◽

Antibody Phage Display ◽

Display Technology

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Production and Characterization of Murine Single Chain Fv Antibodies to Aflatoxin B 1 Derived From a Pre-immunized Antibody Phage Display Library System

Food and Agricultural Immunology ◽

10.1080/0954010021000096373 ◽

2002 ◽

Vol 14 (4) ◽

pp. 255-274 ◽

Cited By ~ 36

Author(s):

STEPHEN DALY ◽

PAUL DILLON ◽

BERNADETTE MANNING ◽

LYNSEY DUNNE ◽

ANTHONY KILLARD ◽

...

Keyword(s):

Phage Display ◽

Phage Display Library ◽

Single Chain ◽

Library System ◽

Single Chain Fv ◽

Antibody Phage ◽

Antibody Phage Display ◽

Aflatoxin B

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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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Selection and characterization of single chain Fv fragments against murine recombinant prion protein from a synthetic human antibody phage display library

Human Antibodies ◽

10.3233/hab-2000-9403 ◽

2000 ◽

Vol 9 (4) ◽

pp. 207-214 ◽

Cited By ~ 8

Author(s):

Estelle Leclerc ◽

Susanne Liemann ◽

Gudrun Wildegger ◽

Stefan W. Vetter ◽

Fredrik Nilsson

Keyword(s):

Phage Display ◽

Prion Protein ◽

Phage Display Library ◽

Human Antibody ◽

Single Chain ◽

Single Chain Fv ◽

Antibody Phage ◽

Antibody Phage Display ◽

Recombinant Prion Protein

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Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein

Viruses ◽

10.3390/v13112201 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2201

Author(s):

Tyng Hwey Tan ◽

Elizabeth Patton ◽

Carol A. Munro ◽

Dora E. Corzo-Leon ◽

Andrew J. Porter ◽

...

Keyword(s):

Therapeutic Potential ◽

Transmembrane Protein ◽

Fluorescent Microscopy ◽

Phage Display Library ◽

Inflammasome Activation ◽

Single Chain ◽

Single Chain Antibody ◽

Surface Plasmon Resonance Analysis ◽

Antibody Phage ◽

Antibody Phage Display

ORF3a has been identified as a viroporin of SARS-CoV-2 and is known to be involved in various pathophysiological activities including disturbance of cellular calcium homeostasis, inflammasome activation, apoptosis induction and disruption of autophagy. ORF3a-targeting antibodies may specifically and favorably modulate these viroporin-dependent pathological activities. However, suitable viroporin-targeting antibodies are difficult to generate because of the well-recognized technical challenge associated with isolating antibodies to complex transmembrane proteins. Here we exploited a naïve human single chain antibody phage display library, to isolate binders against carefully chosen ORF3a recombinant epitopes located towards the extracellular N terminal and cytosolic C terminal domains of the protein using peptide antigens. These binders were subjected to further characterization using enzyme-linked immunosorbent assays and surface plasmon resonance analysis to assess their binding affinities to the target epitopes. Binding to full-length ORF3a protein was evaluated by western blot and fluorescent microscopy using ORF3a transfected cells and SARS-CoV-2 infected cells. Co-localization analysis was also performed to evaluate the “pairing potential” of the selected binders as possible alternative diagnostic or prognostic biomarkers for COVID-19 infections. Both ORF3a N and C termini, epitope-specific monoclonal antibodies were identified in our study. Whilst the linear nature of peptides might not always represent their native conformations in the context of full protein, with carefully designed selection protocols, we have been successful in isolating anti-ORF3a binders capable of recognising regions of the transmembrane protein that are exposed either on the “inside” or “outside” of the infected cell. Their therapeutic potential will be discussed.

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A Single-Chain Antibody Using LoxP511 as the Linker Enables Large-Content Phage Library Construction via Cre/LoxP Recombination

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057114520974 ◽

2014 ◽

Vol 19 (6) ◽

pp. 839-846 ◽

Cited By ~ 1

Author(s):

Yan Zhang ◽

Wei Wang ◽

Ming Lv ◽

Zhou Lin ◽

Jing Geng ◽

...

Keyword(s):

Phage Display ◽

Computer Aided Design ◽

Phage Display Library ◽

Phage Library ◽

Single Chain ◽

Single Chain Antibody ◽

Phage Display Libraries ◽

Antibody Phage ◽

Variable Domains ◽

Aided Design

To obtain natural or “me-better” antibodies (e.g., affinity-maturated antibodies), phage display libraries are widely used. However, the likelihood of obtaining satisfactory antibodies depends on the library content. Here, we used computer-aided design to model the use of the LoxP511 site as a linker between the heavy and light variable domains of an antibody for construction of a large single-chain fragment (scFv) antibody phage library by using the Cre/LoxP recombinant system. Then, we constructed two novel scFvs based on 2C4, namely, AH_scFv15 (15 amino acid [aa] linker; common [SG4]3 sequence) and AH_scFv21 (21-aa linker; LoxP511 sequence), to verify the use of the LoxP511 site as a linker. Our results indicate that LoxP511 could be used effectively for the construction of a large (e.g., 5 × 1012) phage display library of scFv antibodies from which it was possible to isolate an antibody with the same epitope as 2C4 but with higher affinity.

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