Selection of single-chain Fv antibodies against rat C3a and guinea-pig C3a from a large non-immunized phage-library

AbstractFiloviruses, which include ebolaviruses and marburgvirus, can cause outbreaks of highly lethal hemorrhagic fever. This disease causes significant morbidity and mortality in humans and non-human primates, with human fatality rates reaching 90% during some outbreaks. Currently, there are a lack of licensed vaccines or antivirals for these viruses. Since early symptoms of filovirus infection mimic more common diseases, there is a strong unmet public health and biodefense need for broad-spectrum filovirus rapid diagnostics. We have generated a panel of mouse single-chain Fv-antibodies (scFvs) to filovirus glycoproteins (GPs) using cell-free ribosome display and determined their cross-reactivity profiles to all known filovirus species. Two scFvs (4-2 and 22-1) were able to detect all known Ebolavirus and Marburgvirus species. This is the first report on ribosome display scFvs that can detect a broad set of filovirus GPs, which demonstrates their potential use in the development of a new generation of rapid diagnostic immunoassays.

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

Veterinární Medicína ◽

10.17221/5776-vetmed ◽

2012 ◽

Vol 48 (No. 9) ◽

pp. 237-247 ◽

Cited By ~ 9

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.

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Selection of Potential Therapeutic Human Single-Chain Fv Antibodies against Cholecystokinin-B/Gastrin Receptor by Phage Display Technology

BioDrugs ◽

10.1007/s40259-012-0007-0 ◽

2013 ◽

Vol 27 (1) ◽

pp. 55-67 ◽

Cited By ~ 21

Author(s):

Mohammad R. Tohidkia ◽

Farzad Asadi ◽

Jaleh Barar ◽

Yadollah Omidi

Keyword(s):

Phage Display ◽

Single Chain ◽

Phage Display Technology ◽

Gastrin Receptor ◽

Single Chain Fv ◽

Display Technology ◽

Selection Of

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Selection of Rabbit Single-chain Fv Fragments against the Herbicide Atrazine using a New Phage Display System

Food and Agricultural Immunology ◽

10.1080/09540109999870 ◽

1999 ◽

Vol 11 (1) ◽

pp. 5-17 ◽

Cited By ~ 11

Author(s):

Yi Li ◽

WILLIAM COCKBURN ◽

John Kilpatrick ◽

Garry C. Whitelam

Keyword(s):

Phage Display ◽

Display System ◽

Single Chain ◽

Single Chain Fv ◽

Herbicide Atrazine ◽

Selection Of

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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.

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Virus Strain Discrimination Using Recombinant Antibodies

Disease Markers ◽

10.1155/2000/815852 ◽

2000 ◽

Vol 16 (1-2) ◽

pp. 95-97 ◽

Cited By ~ 3

Author(s):

N. Boonham ◽

I. Barker

Keyword(s):

Monoclonal Antibodies ◽

Polyclonal Antibodies ◽

Recombinant Antibody ◽

Plant Viruses ◽

Antibody Fragments ◽

Single Chain ◽

Traditional Methods ◽

Assay Sensitivity ◽

Single Chain Fv ◽

Selection Of

Most routine testing for plant viruses is currently carried out using monoclonal and polyclonal antibodies. Traditional methods of antibody production however can be time consuming and require the use of expensive cell culture facilities. Recombinant antibody technology however is starting to make an impact in this area, enabling the selection of antibody fragments in a few weeks compared with the many months associated with traditional methods and requires only basic microbiological facilities. Single chain Fv antibody fragments (scFv) have been selected from a synthetic phage-antibody library by affinity selection with purifiedPotato virus Y, ordinary strain (PVYO). The scFv selected was specific for PVY and detected 7 out of 9 isolates of PVYOwhilst it did not detect 15 isolates from the closely related necrotic strains PVYNand PVYNTN. In ELISA the scFv could be used to detect virus at concentrations of 50 ng/ml in plant sap and was shown to have similar limits of detection as commercially available PVY monoclonal antibodies. These results highlight the potential of the technology for the selection of strain specific antibodies with an affinity and assay sensitivity similar to traditional monoclonal antibodies and their use in viral diagnostics.

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