scholarly journals Improved Soluble ScFv ELISA Screening Approach for Antibody Discovery Using Phage Display Technology

2017 ◽  
Vol 22 (8) ◽  
pp. 1026-1034 ◽  
Author(s):  
Mohammad R. Tohidkia ◽  
Maryam Sepehri ◽  
Shirin Khajeh ◽  
Jaleh Barar ◽  
Yadollah Omidi
Keyword(s):  
Phage Display ◽  
Recombinant Antibody ◽  
False Negative ◽  
Protein A ◽  
Enzyme Linked Immunosorbent Assay ◽  
Phage Display Technology ◽  
Negative Results ◽  
Specific Phage ◽  
Display Technology ◽  
The Individual

Phage display technology (PDT) is a powerful tool for the isolation of recombinant antibody (Ab) fragments. Using PDT, target molecule-specific phage-Ab clones are enriched through the “biopanning” process. The individual specific binders are screened by the monoclonal scFv enzyme-linked immunosorbent assay (ELISA) that may associate with inevitable false-negative results. Thus, in this study, three strategies were investigated for optimization of the scFvs screening using Tomlinson I and J libraries, including (1) optimizing the expression of functional scFvs, (2) improving the sensitivity of ELISA, and (3) preparing different samples containing scFvs. The expression of all scFv Abs was significantly enhanced when scFv clones were cultivated in the terrific broth (TB) medium at the optimum temperature of 30 °C. The protein A–conjugated with horseradish peroxidase (HRP) was found to be a well-suited reagent for the detection of Ag-bound scFvs in comparison with either anti-c-myc Ab or the mixing procedure. Based on our findings, it seems there is no universal media supplement for an improved expression of all scFvs derived from both Tomlinson I and J libraries. We thus propose that expression of scFv fragments in a microplate scale is largely dependent on a variety of parameters, in particular the scFv clones and relevant sequences.

Characterization of 11-dehydro-thromboxane B2 recombinant antibody obtained by phage display technology

2003 ◽  
Vol 68 (4) ◽  
pp. 273-284 ◽  
Author(s):  
Lilian Rumi Tsuruta ◽  
Yoshihisa Tomioka ◽  
Takanori Hishinuma ◽  
Yoshinori Kato ◽  
Kunihiko Itoh ◽  
...  
Keyword(s):  
Phage Display ◽  
Recombinant Antibody ◽  
Thromboxane B2 ◽  
Phage Display Technology ◽  
Display Technology

scholarly journals Mimotopes for Mycotoxins Diagnosis Based on Random Peptides or Recombinant Antibodies from Phage Library

Molecules ◽  
2021 ◽  
Vol 26 (24) ◽  
pp. 7652
Author(s):  
Wei Sun ◽  
Yan Zhang ◽  
Zhigang Ju
Keyword(s):  
Phage Display ◽  
Recombinant Antibody ◽  
Recombinant Antibodies ◽  
Phage Display Library ◽  
Ease Of Use ◽  
Phage Library ◽  
Phage Display Technology ◽  
Random Peptide ◽  
Display Technology

Mycotoxins, the small size secondary metabolites of fungi, have posed a threat to the safety of medicine, food and public health. Therefore, it is essential to create sensitive and effective determination of mycotoxins. Based on the special affinity between antibody and antigen, immunoassay has been proved to be a powerful technology for the detection of small analytes. However, the tedious preparation and instability of conventional antibodies restrict its application on easy and fast mycotoxins detection. By virtue of simplicity, ease of use, and lower cost, phage display library provides novel choices for antibodies or hapten conjugates, and lead random peptide or recombinant antibody to becoming the promising and environmental friendly immune-reagents in the next generation of immunoassays. This review briefly describes the latest developments on mycotoxins detection using M13 phage display, mainly focusing on the recent applications of phage display technology employed in mycotoxins detection, including the introduction of phage and phage display, the types of phage displayed peptide/recombinant antibody library, random peptides/recombinant antibodies-based immunoassays, as well as simultaneous determination of multiple mycotoxins.

scholarly journals Identification of Novel Single-Domain Antibodies against FGF7 Using Phage Display Technology

2017 ◽  
Vol 23 (2) ◽  
pp. 193-201
Author(s):  
Behzad Jafari ◽  
Maryam Hamzeh-Mivehroud ◽  
Ali A. Moosavi-Movahedi ◽  
Siavoush Dastmalchi
Keyword(s):  
Growth Factor ◽  
Phage Display ◽  
Fibroblast Growth Factor ◽  
Single Domain ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fibroblast Growth ◽  
Phage Display Technology ◽  
Display Technology ◽  
Single Domain Antibodies

Fibroblast growth factor 7 (FGF7) is a member of the fibroblast growth factor (FGF) family of proteins. FGF7 is of stromal origin and produces a paracrine effect on epithelial cells. In the current investigation, we aimed to identify new single-domain antibodies (sdAbs) against FGF7 using phage display technology. The vector harboring the codon-optimized DNA sequence for FGF7 protein was transformed into Escherichia coli BL21 (DE3) pLysS, and then the protein was expressed at the optimized condition. Enzyme-linked immunosorbent assay, circular dichroism spectropolarimetry, and in vitro scratch assay experiments were used to confirm the proper folding and functionality of the purified FGF7 protein. The purity of the produced FGF7 was 92%, with production yield of 3.5 mg/L of culture. Panning against the purified FGF7 was performed, and the identified single-domain antibodies showed significant affinity. Further investigation on one of the selected sdAb displaying phage clones showed concentration-dependent binding to FGF7. The selected sdAb can be used for developing novel tumor-suppressing agents where inhibition of FGF7 is required.

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)

scholarly journals Generation of Fab Fragment-Like Molecular Recognition Proteins against Staphylococcal Enterotoxin B by Phage Display Technology

2010 ◽  
Vol 17 (11) ◽  
pp. 1708-1717 ◽  
Author(s):  
Yuji Urushibata ◽  
Kunihiko Itoh ◽  
Motohiro Ohshima ◽  
Yasuo Seto
Keyword(s):  
Phage Display ◽  
Dissociation Constants ◽  
Staphylococcal Enterotoxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Staphylococcal Enterotoxin B ◽  
Single Chain ◽  
Fab Fragment ◽  
Phage Display Technology ◽  
Display Technology ◽  
Enterotoxin B

ABSTRACT Antigen-binding fragments (Fab fragments) and single-chain variable fragments (scFv) against staphylococcal enterotoxin B (SEB) were produced by phage display technology. SEB epitopes were first identified by phage display approach using the commercial anti-SEB monoclonal antibody ab53981 as the target. Heptamer and dodecamer mimotope peptides recognized by ab53981 were screened from Ph.D-7 or Ph.D-12 random peptide phage libraries expressed in Escherichia coli. The isolated 7-mer and 12-mer mimotopes were shown to share a sequence homologous to 8PDELHK14S in the amino acid sequence of SEB. The N-terminal 15-mer peptide of SEB was determined to be an epitope of ab53981. After immunization of mice with maltose-binding protein-tagged N-terminal 15-mer peptide, a phage display Fab library was constructed using cDNA prepared from the mRNAs of spleen cells. Three phage clones displaying the Fab molecule which recognized SEB were isolated through three rounds of panning. Only one of them produced a soluble Fab fragment from the transformed cells, and the fragment fused with a histidine tag sequence was produced in E. coli cells and converted into scFv. Surface plasmon resonance analysis showed that the dissociation constants of these proteins with SEB were (4.1 ± 1.1) × 10−9 M and (8.4 ± 2.3) × 10−10 M, respectively. The produced molecule was applied to the determination of SEB by enzyme-linked immunosorbent assay and Western blot analysis.

scholarly journals Isolation of recombinant antibody fragments (scFv) by phage display technology for detection of almond allergens in food products

Food Control ◽  
2015 ◽  
Vol 54 ◽  
pp. 322-330 ◽  
Author(s):  
Silvia de la Cruz ◽  
Carolina Cubillos-Zapata ◽  
Inés María López-Calleja ◽  
Satyabrata Ghosh ◽  
Marcos Alcocer ◽  
...  
Keyword(s):  
Phage Display ◽  
Recombinant Antibody ◽  
Food Products ◽  
Antibody Fragments ◽  
Phage Display Technology ◽  
Display Technology ◽  
Recombinant Antibody Fragments

Development and Application of Computational Methods in Phage Display Technology

2020 ◽  
Vol 26 (42) ◽  
pp. 7672-7693 ◽  
Author(s):  
Bifang He ◽  
Anthony Mackitz Dzisoo ◽  
Ratmir Derda ◽  
Jian Huang
Keyword(s):  
Phage Display ◽  
Computational Methods ◽  
Peptide Ligands ◽  
Next Generation ◽  
Phage Display Technology ◽  
Next Generation Sequencing Technology ◽  
Screening Experiments ◽  
Display Technology ◽  
Comprehensive Search ◽  
Generation Sequencing

Background: Phage display is a powerful and versatile technology for the identification of peptide ligands binding to multiple targets, which has been successfully employed in various fields, such as diagnostics and therapeutics, drug-delivery and material science. The integration of next generation sequencing technology with phage display makes this methodology more productive. With the widespread use of this technique and the fast accumulation of phage display data, databases for these data and computational methods have become an indispensable part in this community. This review aims to summarize and discuss recent progress in the development and application of computational methods in the field of phage display. Methods: We undertook a comprehensive search of bioinformatics resources and computational methods for phage display data via Google Scholar and PubMed. The methods and tools were further divided into different categories according to their uses. Results: We described seven special or relevant databases for phage display data, which provided an evidence-based source for phage display researchers to clean their biopanning results. These databases can identify and report possible target-unrelated peptides (TUPs), thereby excluding false-positive data from peptides obtained from phage display screening experiments. More than 20 computational methods for analyzing biopanning data were also reviewed. These methods were classified into computational methods for reporting TUPs, for predicting epitopes and for analyzing next generation phage display data. Conclusion: The current bioinformatics archives, methods and tools reviewed here have benefitted the biopanning community. To develop better or new computational tools, some promising directions are also discussed.

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