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 Generation and Selection of a Panel of Pan-Filovirus Single-Chain Antibodies using Cell-Free Ribosome Display

2018 ◽  
Author(s):  
Adinarayana Kunamneni ◽  
Elizabeth C. Clarke ◽  
Chunyan Ye ◽  
Steven B. Bradfute ◽  
Ravi Durvasula
Keyword(s):  
Hemorrhagic Fever ◽  
Cross Reactivity ◽  
Free Ribosome ◽  
Rapid Diagnostics ◽  
Single Chain ◽  
Ribosome Display ◽  
Single Chain Antibodies ◽  
Single Chain Fv ◽  
New Generation ◽  
Selection Of

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.

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.

scholarly journals Generation of recombinant scFv antibody against Ochratoxin A (OTA)

2018 ◽  
Vol 22 (2) ◽  
pp. 107 ◽  
Author(s):  
Ranya Pranomphon ◽  
Witsanu Srila ◽  
Montarop Yamabhai
Keyword(s):  
Ochratoxin A ◽  
Recombinant Antibody ◽  
Cross Reactivity ◽  
Agricultural Product ◽  
Binding Property ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Recombinant Scfv ◽  
Contaminated Food ◽  
Soluble Scfv

Ochratoxin A (OTA) is a mycotoxin commonly found in agricultural products and can accumulate in the blood and tissues after that consuming contaminated food. Recombinant single-chain antibody fragments (scFv) against OTA were selected from phage display libraries. After of one round of biopanning against BSA-conjugated OTA (OTA-BSA), 52 and 6 phage clones displaying scFv antibodies were isolated from human (Yamo I.3) and rabbit (Bozmix I.2) libraries. Two phage clones (one from each libraries, i.e., yOTA1e3 and bOTA2a9) showed binding to free toxin by competitive ELISA. The soluble scFv antibodies were produced by superinfecting phage clones into E. coli suppressor strain HB2151. The scFv genes from these two phage clones were sub-cloned into pKP300ΔIII vectors to generate scFv-AP fusions. The binding affinity (IC50) of antibody derived from human library was higher than those from rabbit library. The binding property of recombinant antibody in the form of scFv-AP was better than those of soluble scFv form. Cross-reactivity analysis indicated that the two recombinant antibodies did not cross-react with other soluble toxins, namely AFB1, DON, ZEN and FB. The ability to use the recombinant scFv-AP to detect contaminated toxins in agricultural product (corn) was demonstrated.

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

1998 ◽  
Vol 35 (6-7) ◽  
pp. 365
Author(s):  
J.-H. Pütz ◽  
T. Vaughan ◽  
W. Roake ◽  
E. Nagel ◽  
A. Meyer zu Vilsendorl ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Phage Library ◽  
Single Chain ◽  
Single Chain Fv ◽  
Selection Of

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

In Vitro Shoot Regeneration from Callus Derived from Marubakaido Apple Rootstock under Different Aluminium Concentrations

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 460e-460 ◽  
Author(s):  
Marisa F. de Oliveira ◽  
Gerson R. de L. Fortes ◽  
João B. da Silva
Keyword(s):  
Shoot Regeneration ◽  
Dichlorophenoxyacetic Acid ◽  
Apple Rootstock ◽  
Under Five ◽  
Significant Difference ◽  
Previously Treated ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
In Vitro Shoot Regeneration

The aim of this work was to evaluate the organogenesis of Marubakaido apple rootstock under different aluminium concentratons. The explants were calli derived from apple internodes treated with either 2,4-dichlorophenoxyacetic acid or pichloram at 0.5 and 1.0 μM and under five different aluminium concentrations (0, 5, 10, 15, 20 mg/L). These calli were then treated with aluminium at 0, 5, 10, 15, and 20 mg/L. It was observed shoot regeneration only for those calli previously treated with pichloram. There were no significant difference among the aluminium concentrations.

Characterization of the Reproductive Mode in Guayule In Vitro

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 483a-483
Author(s):  
Roy N. Keys ◽  
Dennis T. Ray ◽  
David A. Dierig
Keyword(s):  
Field Experiments ◽  
Reproductive Mode ◽  
Dichlorophenoxyacetic Acid ◽  
Initial Field ◽  
Breeding Lines ◽  
Reproductive Modes ◽  
Desert Southwest ◽  
2,4 Dichlorophenoxyacetic Acid

Guayule (Parthenium argentatum Gray, Asteraceae) is a latex-producing perennial desert shrub that is potentially of economic importance as an industrial crop for the desert Southwest. It is known to possess complex reproductive modes. Diploids are predominantly sexual and self-incompatible, while polyploids show a range of apomictic potential and self-compatibility. This paper describes the development of a relatively rapid and simple technique for characterizing reproductive modes of breeding lines of P. argentatum. Initial field experiments were based on an auxin test used successfully to characterize reproductive mode in the Poaceae. The application of 2,4-dichlorophenoxyacetic acid inhibited embryo formation in P. argentatum, but this was not the case with other auxins tested. Results of field experiments were ambiguous because: 1) the floral structure of P. argentatum is such that auxins might not have penetrated to the ovules, and 2) there was potential self-fertilization by pollen released within isolation bags. Therefore, in vitro culture of flower heads was tested because it provided much better control of environmental conditions, growth regulator application, and pollen release. Auxin alone, or in combination with gibberellic acid or kinetin, inhibited parthenogenesis in vitro. Embryo production did not vary using two substantially different nutrient media. In vitro flower head culture using a (Nitsch and Nitsch) liquid nutrient medium without growth regulators, enabled characterization of the reproductive mode of seven breeding lines, ranging from predominantly sexual to predominantly apomictic. The results of this technique were substantiated using RAPD analyzes of progeny arrays from controlled crosses.

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