scholarly journals Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of the Pseudonitzschia pungens Toxin Domoic Acid

2006 ◽  
Vol 72 (5) ◽  
pp. 3343-3349 ◽  
Author(s):  
William J. J. Finlay ◽  
Iain Shaw ◽  
Joanna P. Reilly ◽  
Marian Kane
Keyword(s):  
Domoic Acid ◽  
Recombinant Antibody ◽  
Variable Region ◽  
Antibody Fragments ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Antibody ◽  
High Affinity ◽  
Single Chain Fv ◽  
Recombinant Antibody Fragments

ABSTRACT Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single VH and VL genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.

Construction and Expression of a Bifunctional Single-Chain Antibody against Bacillus cereusSpores

1998 ◽  
Vol 64 (7) ◽  
pp. 2490-2496 ◽  
Author(s):  
Kai Koo ◽  
Peggy M. Foegeding ◽  
Harold E. Swaisgood
Keyword(s):  
Monoclonal Antibody ◽  
Recombinant Antibody ◽  
Expression System ◽  
Variable Region ◽  
Hybridoma Cells ◽  
Antigen Specificity ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Western Blots ◽  
Variable Region Genes

ABSTRACT The variable-region genes of monoclonal antibody againstBacillus cereus spores were cloned from mouse hybridoma cells by reverse transcription-PCR. The heavy- and light-chain variable-region genes were connected by a 45-base linker DNA to allow folding of the fusion protein into a functional tertiary structure. For detection of protein expression, a 10-amino-acid strep tag (biotin-like peptide) was attached to the C terminus of recombinant antibody as the reporter peptide. The single-chain antibody construct was inserted into the expression vector and expressed in Escherichia coliunder the control of the T7 RNA polymerase-T7 promoter expression system. The expressed single-chain antibody was detected on Western blots by using a streptavidin-conjugated enzyme system. This small recombinant antibody fragment (ca. 28,000 Da by calculation) hadB. cereus spore binding ability and antigen specificity similar to those of its parent native monoclonal antibody.

scholarly journals High-Affinity Recombinant Antibody Fragments (Fabs) Can Be Applied in Peptide Enrichment Immuno-MRM Assays

2014 ◽  
Vol 13 (4) ◽  
pp. 2187-2196 ◽  
Author(s):  
Jeffrey R. Whiteaker ◽  
Lei Zhao ◽  
Christian Frisch ◽  
Francisco Ylera ◽  
Stefan Harth ◽  
...  
Keyword(s):  
Recombinant Antibody ◽  
Antibody Fragments ◽  
High Affinity ◽  
Recombinant Antibody Fragments ◽  
Peptide Enrichment

scholarly journals Virus Strain Discrimination Using Recombinant Antibodies

2000 ◽  
Vol 16 (1-2) ◽  
pp. 95-97 ◽  
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.

scholarly journals Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water

2002 ◽  
Vol 68 (11) ◽  
pp. 5288-5295 ◽  
Author(s):  
Jacqui McElhiney ◽  
Mathew Drever ◽  
Linda A. Lawton ◽  
Andy J. Porter
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Phage Display ◽  
High Performance ◽  
Recombinant Antibody ◽  
World Health ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Cyanobacterial Toxin

ABSTRACT A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 μg liter−1) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 μg of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.

scholarly journals Expression, purification and characterization of α-synuclein fibrillar specific scFv from inclusion bodies

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0241773
Author(s):  
Vijay Gupta ◽  
Indulekha P. Sudhakaran ◽  
Zeyaul Islam ◽  
Nishant N. Vaikath ◽  
Issam Hmila ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Recombinant Antibody ◽  
Structural Alignment ◽  
Lewy Bodies ◽  
High Specificity ◽  
Binding Pocket ◽  
Antibody Fragments ◽  
Structural Topology ◽  
Single Chain ◽  
Recombinant Antibody Fragments

Aggregation of α-synuclein (α-syn) has been implicated in multiple neurodegenerative disorders including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), collectively grouped as synucleinopathies. Recently, recombinant antibody fragments (Fab, scFvs and diabodies) against α-syn have emerged as an alternative to the traditional full-length antibody in immunotherapeutic approaches owing to their advantages including smaller size and higher stability, specificity and affinity. However, most of the recombinant antibody fragments tend to be expressed as inclusion bodies (IBs) making its purification extremely challenging. In the current study, a single-chain variable fragment (scFv-F) antibody, targeting the pathogenic α-syn fibrils, was engineered and expressed in E. coli. Majority of the expressed scFv-F accumulated in insoluble aggregates as IBs. A variety of mild and harsh solubilizing conditions were tested to solubilize IBs containing scFv-F to obtain the active protein. To preserve secondary structure and bioactivity, a mild solubilizing protocol involving 100 mM Tris, pH 12.5 with 2 M urea was chosen to dissolve IBs. Slow on-column refolding method was employed to subsequently remove urea and obtain active scFv-F. A three-dimensional (3D) model was built using homology modeling and subjected to molecular docking with the known α-syn structure. Structural alignment was performed to delineate the potential binding pocket. The scFv-F thus purified demonstrated high specificity towards α-syn fibrils compared to monomers. Molecular modeling studies suggest that scFv-F shares the same structural topology with other known scFvs. We present evidence through structural docking and alignment that scFv-F binds to α-syn C-terminal region. In conclusion, mild solubilization followed by slow on-column refolding can be utilized as a generalized and efficient method for hard to purify disease relevant insoluble proteins and/or antibody molecules from IBs.

Expression, purification and characterization of α-synuclein fibrillar specific scFv from inclusion bodies

2020 ◽  
Author(s):  
Vijay Gupta ◽  
Indulekha P. Sudhakaran ◽  
Zeyaul Islam ◽  
Nishant N. Vaikath ◽  
Issam Hmila ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Recombinant Antibody ◽  
Structural Alignment ◽  
Lewy Bodies ◽  
High Specificity ◽  
Binding Pocket ◽  
Antibody Fragments ◽  
Structural Topology ◽  
Single Chain ◽  
Recombinant Antibody Fragments

Abstract Background: Aggregation of α-synuclein (α-syn) has been implicated in multiple neurodegenerative disorders including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), collectively grouped as synucleinopathies. Recently, recombinant antibody fragments (Fab, scFvs and diabodies) against α-syn have emerged as an alternative to the traditional full-length antibody in immunotherapeutic approaches owing to their advantages including smaller size and higher stability, specificity and affinity. However, most of the recombinant antibody fragments tend to be expressed as inclusion bodies (IBs) making its purification extremely challenging. Results: In the current study, a single-chain variable fragment (scFv-F) antibody, targeting the pathogenic α-syn fibrils, was engineered and expressed in E.coli. Majority of the expressed scFv-F accumulated in insoluble aggregates as IBs. A variety of mild and harsh solubilizing conditions were tested to solubilize IBs containing scFv-F to obtain the active protein. To preserve secondary structure and bioactivity, a mild solubilizing protocol involving 100 mM Tris, pH 12.5 with 2 M urea was chosen to dissolve IBs. Slow on-column refolding method was employed to subsequently remove urea and obtain active scFv-F. A three-dimensional (3D) model was built using homology modeling and subjected to molecular docking with the known α-syn structure. Structural alignment was performed to delineate the potential binding pocket. Conclusion: The scFv-F thus purified demonstrated high specificity towards α-syn fibrils compared to monomers. Molecular modeling studies suggest that scFv-F shares the same structural topology with other known scFvs. We present evidence through structural docking and alignment that scFv-F binds to α-syn C-terminal region. In conclusion, mild solubilization followed by slow on-column refolding can be utilized as a generalized and efficient method for hard to purify disease relevant insoluble proteins and/or antibody molecules from IBs.

scholarly journals Optimal Neutralization of Centruroides noxius Venom Is Understood through a Structural Complex between Two Antibody Fragments and the Cn2 Toxin

2015 ◽  
Vol 291 (4) ◽  
pp. 1619-1630 ◽  
Author(s):  
Lidia Riaño-Umbarila ◽  
Luis M. Ledezma-Candanoza ◽  
Hugo Serrano-Posada ◽  
Guillermo Fernández-Taboada ◽  
Timoteo Olamendi-Portugal ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Recombinant Antibody ◽  
Current Trend ◽  
Cross Reactivity ◽  
Antibody Fragments ◽  
Crystallographic Structure ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Neutralizing Capacity ◽  
Centruroides Noxius

The current trend of using recombinant antibody fragments in research to develop novel antidotes against scorpion stings has achieved excellent results. The polyclonal character of commercial antivenoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals, we aimed to develop an equivalent recombinant antivenom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpion Centruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously poisoned animals. The crystallographic structure of the ternary complex scFv LR-Cn2-scFv RU1 allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom.

Phage-displayed recombinant single-chain antibody fragments with high affinity for cholesteryl ester transfer protein (CETP): cDNA cloning, characterization and CETP quantification

2004 ◽  
Vol 42 (3) ◽  
Author(s):  
Andreas Ritsch ◽  
Christoph Ebenbichler ◽  
Elisabeth Naschberger ◽  
Wilfried Schgoer ◽  
Ursula Stanzl ◽  
...  
Keyword(s):  
Cholesteryl Ester ◽  
Cholesteryl Ester Transfer Protein ◽  
Density Lipoprotein ◽  
Phage Display Library ◽  
Antibody Fragments ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Recombinant Phage ◽  
Transfer Protein ◽  
High Affinity

AbstractCholesteryl ester transfer protein (CETP) greatly affects the metabolism of all lipoprotein classes including low-density lipoprotein (LDL) and high-density lipoprotein (HDL), bothknown to constitute powerful risk factors for coronary artery disease (CAD). We now report the successful first cloning and characterization of single-chain antibody fragments specific for CETP. A recombinant phage display library was generated using spleen mRNA isolated from BALB/c mice that had been immunized with highly purified CETP. Screening of the library yielded two single-chain antibody fragments with high affinity for CETP, termed 1CL8 and 1CL10, displaying respective K

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