Type

Journal Article

Authors

Richard O'Kennedy
Martin Danaher
Elaine Darcy
Paul Leonard
Jenny Fitzgerald

Subjects

Microbiology

Topics
antibody specificity peptide library antigens enzyme linked immunosorbent assay piperidines calibration single chain antibodies quinazolinones humans halofuginone limit of detection genetics immunology

Light-chain shuffling from an antigen-biased phage pool allows 185-fold improvement of an anti-halofuginone single-chain variable fragment. (2010)

Abstract Halofuginone is an antiprotozoal drug used in the treatment of coccidiosis in poultry, a contagious enteric disease caused by parasites of the Eimeria spp. To ensure that food is free from any halofuginone residues and safe for human consumption, a rapid method to detect these residues below the maximum residue limits (MRLs) in a variety of matrices is necessary. To address this need, we constructed an immune single-chain variable fragment (scFv) library from the RNA of a halofuginone-immunized chicken and selected halofuginone-specific scFv by phage display. The best clone isolated from the library had a limit of detection of 30 ng/ml as determined by enzyme-linked immunosorbent assay (ELISA). However, the minimum MRL for halofuginone in certain foodstuffs can be as low as 1 ng/ml, well below the sensitivity of the selected antibody. The selected antibody was then affinity maturated by light-chain shuffling to further improve the antibody's assay performance. The halofuginone-specific heavy-chain pool of the biopanned library was assembled with the light-chain repertoire amplified from the original prepanned library. This resulted in a heavy-chain-biased library from which an scFv with the potential to detect halofuginone residues as low as 80 pg/ml was isolated, a 185-fold improvement over the original scFv. This new chain-shuffled scFv was incorporated into a validated ELISA (according to Commission Regulation 2002/657/EC) for the sensitive detection of halofuginone in spiked processed egg samples.
Collections Ireland -> Dublin City University -> PubMed

Full list of authors on original publication

Richard O'Kennedy, Martin Danaher, Elaine Darcy, Paul Leonard, Jenny Fitzgerald

Experts in our system

1
R O'Kennedy
Dublin City University
Total Publications: 197
 
2
Martin Danaher
Teagasc
Total Publications: 58
 
3
Elaine Darcy
Dublin City University
Total Publications: 6
 
4
Paul Leonard
Dublin City University
Total Publications: 27
 
5
Jenny Fitzgerald
Dublin City University
Total Publications: 18