PMID- 21078281
OWN - NLM
STAT- MEDLINE
DCOM- 20110419
LR  - 20121115
IS  - 1096-0309 (Electronic)
IS  - 0003-2697 (Linking)
VI  - 410
IP  - 1
DP  - 2011 Mar 1
TI  - Light-chain shuffling from an antigen-biased phage pool allows 185-fold 
      improvement of an anti-halofuginone single-chain variable fragment.
PG  - 27-33
LID - 10.1016/j.ab.2010.11.009 [doi]
AB  - 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.
CI  - 2010 Elsevier Inc. All rights reserved.
FAU - Fitzgerald, Jenny
AU  - Fitzgerald J
AD  - School of Biotechnology and National Centre for Sensor Research, Dublin City 
      University, Dublin 9, Ireland.
FAU - Leonard, Paul
AU  - Leonard P
FAU - Darcy, Elaine
AU  - Darcy E
FAU - Danaher, Martin
AU  - Danaher M
FAU - O'Kennedy, Richard
AU  - O'Kennedy R
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20101113
PL  - United States
TA  - Anal Biochem
JT  - Analytical biochemistry
JID - 0370535
RN  - 0 (Antigens)
RN  - 0 (Peptide Library)
RN  - 0 (Piperidines)
RN  - 0 (Quinazolinones)
RN  - 0 (Single-Chain Antibodies)
RN  - L31MM1385E (halofuginone)
SB  - IM
MH  - Antibody Specificity
MH  - Antigens/*immunology
MH  - Calibration
MH  - Enzyme-Linked Immunosorbent Assay
MH  - Humans
MH  - Limit of Detection
MH  - *Peptide Library
MH  - Piperidines/analysis/*immunology
MH  - Quinazolinones/analysis/*immunology
MH  - Single-Chain Antibodies/*genetics/*immunology
EDAT- 2010/11/17 06:00
MHDA- 2011/04/20 06:00
CRDT- 2010/11/17 06:00
PHST- 2010/06/29 00:00 [received]
PHST- 2010/10/06 00:00 [revised]
PHST- 2010/11/04 00:00 [accepted]
PHST- 2010/11/17 06:00 [entrez]
PHST- 2010/11/17 06:00 [pubmed]
PHST- 2011/04/20 06:00 [medline]
AID - S0003-2697(10)00722-0 [pii]
AID - 10.1016/j.ab.2010.11.009 [doi]
PST - ppublish
SO  - Anal Biochem. 2011 Mar 1;410(1):27-33. doi: 10.1016/j.ab.2010.11.009. Epub 2010 
      Nov 13.