Selection of a Single Chain Variable Fragment Antibody against Ivermectin from a Phage Displayed Library

Abstract Background and Objective: Overexpression of the EGFR, from the ErbB receptor family, has been observed in several cancers and causes resistance to therapeutic antibodies, such as Herceptin. In this study, we produced a recombinant single-chain variable fragment (scFv) antibody against the EGFR dimerization domain. Methods: The recombinant scFv was generated using a cell-based subtractive panning strategy. Subtractive panning was performed on genetically engineered VERO/EGFR cells and cancerous MDA-MB-468 cells. Phage cell-ELISA was used to monitor the binding of the selected scFvs to the dimerization domain of EGFR. Inhibition of EGFR and HER2 dimerization by the produced scFvs were finally evaluated using the dimerization inhibition test. Results: PCR fingerprinting results showed a uniform digestion pattern following the third round of panning that confirmed the success of subtractive panning. Moreover, cell-ELISA validated the reactivity of the produced scFvs to EGFR after stimulation with EGF. The dimerization inhibition test showed the capacity of the scFvs to inhibit EGFR and HER2 dimerization. Conclusions: Directed HER2 targeting showed to be more effective to target the functional domain of the cell receptor for the complete blockade of the intracellular signaling pathway. The subtractive panning strategy used in this study could control the process of directed selection of specific antibodies against the dimerization domain of EGFR. The selected antibodies might then be functionally tested for antitumor effects in both in vitro and in vivo studies.

Download Full-text

Phage antibody library screening for the selection of novel high-affinity human single-chain variable fragment against gastrin receptor: an in silico and in vitro study

DARU Journal of Pharmaceutical Sciences ◽

10.1007/s40199-018-0233-1 ◽

2019 ◽

Vol 27 (1) ◽

pp. 21-34 ◽

Cited By ~ 6

Author(s):

Sepideh Jalilzadeh-Razin ◽

Malihe Mantegi ◽

Mohammad R. Tohidkia ◽

Yaghub Pazhang ◽

Mohammad M. Pourseif ◽

...

Keyword(s):

In Silico ◽

In Vitro Study ◽

Library Screening ◽

Single Chain Variable Fragment ◽

Antibody Library ◽

Single Chain ◽

High Affinity ◽

Phage Antibody Library ◽

Selection Of

Download Full-text

Selection of a Single Chain Variable Fragment Antibody (scFv) against Subtilisin BRC and its Interaction with Subtilisin BRC

Current Biotechnology ◽

10.2174/2211550108666190417113342 ◽

2019 ◽

Vol 8 (1) ◽

pp. 24-31

Author(s):

Chol-Jin Kim ◽

Sunll Choe ◽

Kum-Chol Ri ◽

Chol-Ho Kim ◽

Hyon-Gwang Li ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Phage Display ◽

Affinity Purification ◽

Phage Library ◽

Amino Acid Residues ◽

Single Chain Variable Fragment ◽

Single Chain ◽

E Coli ◽

Selection Of

Background: The focus of this study was the selection of a single chain variable fragment antibody (scFv) against subtilisin BRC, a fibrinolytic enzyme using phage display, and to characterize the interaction between the antibody and subtilisin BRC. Methods: The subtilisin BRC-specific phage clones were selected using Griffin.1 scFv phage library and sequenced. The gene of subtilisin BRC-specific scFv (scFv-BRC) from selected phage clone was expressed in E. coli and scFv-BRC was characterized. Molecular modeling of the three-dimensional (3D) structures of scFv-BRC was performed using MODELLER 9.19 modeling software and assessed by PROCHEK. Molecular docking of subtilisin BRC with scFv-BRC was carried out using PATCHDOCK. Results: The size of scFv-BRC gene is 635bp and it consists of 54bp of heavy chain region (VH), 336bp of light chain region (VL), 45bp of a linker. scFv-BRC was actively expressed by E. coli expression vector pET28a-scFv in E. coli BL21 (DE3), and the amount of expressed scFv-BRC was about 50 mg/L. Its molecular weight is ~26kDa. The CDR domain of scFv-BRC consists of 6 amino acids in CDR L1, 3 amino acids in CDR L2 and 9 amino acids in CDR L3. Docking results of subtilisin BRC and scFv-BRC showed global energy of - 56.29 kJ/mol. Furthermore, the results showed that amino acid residues in subtilisin BRC for binding with scFv-BRC are Tyr6, Ser182, Ser204, and Gln206. Conclusion: scFv against subtilisin BRC selected using phage display showed relatively strong binding energy with subtilisin BRC. The amino acid residues in subtilisin BRC for binding with scFv-BRC are not relevant to that in subtilisin BRC for binding with its substrates. These results suggested that scFv-BRC can be used as a ligand for detection and affinity purification of subtilisin BRC.

Download Full-text

Selection of single-chain variable fragment antibodies against fenitrothion by ribosome display

Analytical Biochemistry ◽

10.1016/j.ab.2011.10.044 ◽

2012 ◽

Vol 421 (1) ◽

pp. 130-137 ◽

Cited By ~ 8

Author(s):

Yihui Luo ◽

Yuxian Xia

Keyword(s):

Single Chain Variable Fragment ◽

Single Chain ◽

Ribosome Display ◽

Selection Of

Download Full-text

A Cell-Based Subtractive Panning Strategy for Selection of Conformation-Specific Single-Chain Variable-Fragment (Scfv) Against Dimerization Domain of EGFR

10.21203/rs.3.rs-421317/v1 ◽

2021 ◽

Author(s):

Reza Valadan ◽

Mina Dabiri ◽

Mohsen Tehrani ◽

Gholamreza Hashemi Tabar ◽

Alireza Rafiei

Keyword(s):

Inhibition Test ◽

Functional Domain ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Dimerization Domain ◽

Antitumor Effects ◽

Egfr Inhibition ◽

A Cell ◽

Cell Elisa ◽

Selection Of

Abstract Background and Objective: Overexpression of the EGFR, from the ErbB receptor family, has been observed in several cancers and causes resistance to therapeutic antibodies, such as Herceptin. In this study, we produced a recombinant single-chain variable fragment (scFv) antibody against the EGFR dimerization domain. Methods The recombinant scFv was generated using a cell-based subtractive panning strategy. Subtractive panning was performed on genetically engineered VERO/EGFR cells and cancerous MDA-MB-468 cells. Phage cell-ELISA was used to monitor the binding of the selected scFvs to the dimerization domain of EGFR. Inhibition of EGFR and HER2 dimerization by the produced scFvs were finally evaluated using the dimerization inhibition test. Results PCR fingerprinting results showed a uniform digestion pattern following the third round of panning that confirmed the success of subtractive panning. Moreover, cell-ELISA validated the reactivity of the produced scFvs to EGFR after stimulation with EGF. The dimerization inhibition test showed the capacity of the scFvs to inhibit EGFR and HER2 dimerization. Conclusions Directed HER2 targeting showed to be more effective to target the functional domain of the cell receptor for the complete blockade of the intracellular signaling pathway. The subtractive panning strategy used in this study could control the process of directed selection of specific antibodies against the dimerization domain of EGFR. The selected antibodies might then be functionally tested for antitumor effects in both in vitro and in vivo studies.

Download Full-text

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

Download Full-text

Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed inE. coli

mAbs ◽

10.4161/mabs.28635 ◽

2014 ◽

Vol 6 (4) ◽

pp. 1084-1093 ◽

Cited By ~ 1

Author(s):

Adriano Podestà ◽

Serena Rossi ◽

Ilaria Massarelli ◽

Sara Carpi ◽

Barbara Adinolfi ◽

...

Keyword(s):

Phage Library ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Ache Inhibitors ◽

Selection Of ◽

Human Butyrylcholinesterase

Download Full-text

Efficient silkworm expression of a single-chain variable fragment antibody against ginsenoside Re and its application in ELISA for quality control of total ginsenosides in various ginseng

Planta Medica ◽

10.1055/s-0030-1264815 ◽

2010 ◽

Vol 76 (12) ◽

Author(s):

S Sakamoto ◽

B Pongkitwitoon ◽

S Nakamura ◽

K Maenaka ◽

H Tanaka ◽

...

Keyword(s):

Quality Control ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Ginsenoside Re

Download Full-text

Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.64.12.4862-4869.1998 ◽

1998 ◽

Vol 64 (12) ◽

pp. 4862-4869 ◽

Cited By ~ 39

Author(s):

Jörg F. Rippmann ◽

Michaela Klein ◽

Christian Hoischen ◽

Bodo Brocks ◽

Wolfgang J. Rettig ◽

...

Keyword(s):

Escherichia Coli ◽

Proteus Mirabilis ◽

Binding Activity ◽

Host Cells ◽

Heterologous Proteins ◽

Single Chain Variable Fragment ◽

Single Chain ◽

E Coli ◽

Active Product ◽

Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

Download Full-text