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

mAbs ◽  
2014 ◽  
Vol 6 (4) ◽  
pp. 1084-1093 ◽  
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

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

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.

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

2010 ◽  
Vol 58 (9) ◽  
pp. 5387-5391 ◽  
Author(s):  
Shuang Wen ◽  
Xiao Zhang ◽  
Yuan Liu ◽  
Qingqing Zhang ◽  
Xianjin Liu ◽  
...  
Keyword(s):  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Selection Of

scholarly journals A Cell-Based Subtractive Panning Strategy for Selection of Conformation-Specific Single-Chain Variable-Fragment (scFv) against Dimerization Domain of EGFR

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.

scholarly journals Production of scFv recombinant fragments against 2,4-dichlorophenoxyacetic acid hapten using naďve phage library

2012 ◽  
Vol 48 (No. 9) ◽  
pp. 237-247 ◽  
Author(s):  
J. Brichta ◽  
H. Vesela ◽  
M. Franek
Keyword(s):  
Cross Reactivity ◽  
Phage Library ◽  
Dichlorophenoxyacetic Acid ◽  
Single Chain ◽  
Metal Affinity ◽  
Recombinant Scfv ◽  
Storage Buffer ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
Recombinant Fragments ◽  
Selection Of

Three single chain variable fragment (scFv) antibodies against 2,4-dichlophenoxyacetic acid (2,4-D) herbicide were produced by the Griffin1.library. The selection of the scFv from the phage library was carried out by 2,4-D-protein coated tubes with different levels of hapten substitution in the conjugate. The scFv phage clones were isolated within the five round library panning and the antibodies were expressed in Escherichia coli HB2151. The recombinant products were purified by metal affinity chromatography yielding 200 g of pure scFv per 1 liter of bacterial culture. The antibody fragments provided steep curves in conventional indirect ELISA having the IC<sub>50</sub> values from 10.2 to 14.5 ng/ml established for 2,4-D standard. Interestingly enough, the recombinant ScFv E1 antibody exhibited 68% cross-reactivity with 2,4-dichlorphenol (2,4-D = 100%), and 38.0% with methylchlorophenoxyacetic acid (MCPA) whereas reaction with other phenoxyacetic compounds was low. Similar characteristics were obtained for other two recombinant products. Low stability for the isolated scFv antibodies was found in storage buffer even in the presence of stabilizers and protease inhibitors. Factors influencing stability of the recombinant antibodies are discussed.

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

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.

Selection of single chain variable fragment (scFv) antibodies from a hyperimmunized phage display library for the detection of the antibiotic monensin

2010 ◽  
Vol 360 (1-2) ◽  
pp. 103-118 ◽  
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
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