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

2014 ◽  
Vol 19 (6) ◽  
pp. 839-846 ◽  
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.

Hevein-specific recombinant IgE antibodies from human single-chain antibody phage display libraries

2003 ◽  
Vol 278 (1-2) ◽  
pp. 271-281 ◽  
Author(s):  
Marja-Leena Laukkanen ◽  
Soili Mäkinen-Kiljunen ◽  
Kirsi Isoherranen ◽  
Tari Haahtela ◽  
Hans Söderlund ◽  
...  
Keyword(s):  
Phage Display ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Ige Antibodies ◽  
Phage Display Libraries ◽  
Antibody Phage ◽  
Antibody Phage Display

Single chain variable fragment antibodies against Shiga toxins isolated from a human antibody phage display library

Vaccine ◽  
2011 ◽  
Vol 29 (33) ◽  
pp. 5340-5346 ◽  
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

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

BioTechniques ◽  
2007 ◽  
Vol 42 (6) ◽  
pp. 760-765 ◽  
Author(s):  
Bizhi Shi ◽  
Huamao Wang ◽  
Shengrong Guo ◽  
Yuhong Xu ◽  
Zonghai Li ◽  
...  
Keyword(s):  
Phage Display ◽  
Bacterial Cells ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Helper Phage ◽  
Antibody Phage ◽  
Antibody Phage Display

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

Leukemia ◽  
1998 ◽  
Vol 12 (8) ◽  
pp. 1295-1302 ◽  
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

scholarly journals Phage display and kinetic selection of antibodies that specifically inhibit amyloid self-replication

2017 ◽  
Vol 114 (25) ◽  
pp. 6444-6449 ◽  
Author(s):  
Anna Munke ◽  
Jonas Persson ◽  
Tanja Weiffert ◽  
Erwin De Genst ◽  
Georg Meisl ◽  
...  
Keyword(s):  
Phage Display ◽  
Amyloid Fibrils ◽  
Amyloid Β ◽  
Amyloid Β Peptide ◽  
Nucleation Process ◽  
Single Chain ◽  
Secondary Nucleation ◽  
Single Chain Antibody ◽  
Phage Display Libraries ◽  
Selection Of

The aggregation of the amyloid β peptide (Aβ) into amyloid fibrils is a defining characteristic of Alzheimer’s disease. Because of the complexity of this aggregation process, effective therapeutic inhibitors will need to target the specific microscopic steps that lead to the production of neurotoxic species. We introduce a strategy for generating fibril-specific antibodies that selectively suppress fibril-dependent secondary nucleation of the 42-residue form of Aβ (Aβ42). We target this step because it has been shown to produce the majority of neurotoxic species during aggregation of Aβ42. Starting from large phage display libraries of single-chain antibody fragments (scFvs), the three-stage approach that we describe includes (i) selection of scFvs with high affinity for Aβ42 fibrils after removal of scFvs that bind Aβ42 in its monomeric form; (ii) ranking, by surface plasmon resonance affinity measurements, of the resulting candidate scFvs that bind to the Aβ42 fibrils; and (iii) kinetic screening and analysis to find the scFvs that inhibit selectively the fibril-catalyzed secondary nucleation process in Aβ42 aggregation. By applying this approach, we have identified four scFvs that inhibit specifically the fibril-dependent secondary nucleation process. Our method also makes it possible to discard antibodies that inhibit elongation, an important factor because the suppression of elongation does not target directly the production of toxic oligomers and may even lead to its increase. On the basis of our results, we suggest that the method described here could form the basis for rationally designed immunotherapy strategies to combat Alzheimer’s and related neurodegenerative diseases.

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

Viruses ◽  
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.

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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