title: Light-chain shuffling from an antigen-biased phage pool allows 185-fold improvement of an anti-halofuginone single-chain variable fragment.
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.
authors.affiliation: School of Biotechnology and National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland.
authors.forename: Jenny
authors.initials: J
authors.lastname: Fitzgerald
authors.forename: Paul
authors.initials: P
authors.lastname: Leonard
authors.forename: Elaine
authors.initials: E
authors.lastname: Darcy
authors.forename: Martin
authors.initials: M
authors.lastname: Danaher
authors.forename: Richard
authors.initials: R
authors.lastname: O'Kennedy
mesh_terms: D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology*; D002138:Calibration; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D057230:Limit of Detection; D019151:Peptide Library; D010880:Piperidines / Q000032:analysis / Q000276:immunology*; D052999:Quinazolinones / Q000032:analysis / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology*
chemical_list: D000941:Antigens; D019151:Peptide Library; D010880:Piperidines; D052999:Quinazolinones; D057127:Single-Chain Antibodies; C010176:halofuginone