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ELISA test kits for AOZ and AMOZ (metabolites of furazolidone and furaltadone, capable of detecting these drugs at concentrations below the EU minimum required performance limit

Furazolidone, a nitrofuran antibiotic, is banned from use in food animal production within the European Union. Increasingly, compliance with this ban is monitored by use of analytical methods to detect a stable tissue-bound metabolite, 3-amino-2-oxazolidinone (AOZ). Widespread use of furazolidone in poultry and prawns imported into Europe highlighted the urgent need for development of nitrofuran immunoassay screening tests. Polyclonal antibodies were produced to detect AOZ, following derivatisation with o-nitrobenzaldehyde. A carboxyphenyl derivative of AOZ was prepared, purified and conjugated to immunogenic carrier protein. Six antisera were produced from the immunisation of seven rabbits using various immunogen doses and time-scales. IC50 values, as determined by competitive enzyme-linked immunosorbent assay (ELISA) suggested that reducing immunogen dose from 0.3 to 0.05 mg, while lengthening rest periods between booster immunisations from 2 to 8 weeks, increased the sensitivity of the antibodies to
3-{[(2-nitrophenyl)methylene]amino}-2-oxazolidinone (NPAOZ) from 3.8 to 0.3 µg.l−1. An IC50 of 0.065 µg.l−1 (AOZ in the form of NPAOZ) was achieved with antiserum R670 by altering ELISA conditions. This antibody was highly specific for NPAOZ and did not cross-react with various nitrofuran metabolites, their nitrophenyl derivatives or a range of veterinary drugs. Antibody R670 is suitable for incorporation into an immunoassay for AOZ with sufficient sensitivity to satisfy current criteria for monitoring of veterinary drug residues. A similar procedure was used to produce antisera against AMOZ the equivalent metabolite of furaltadone.

Both assays were developed into commercial ELISA test kits, which were launched by the FoodBRAND team during 2003, in response to the growing international need for testing procedures for the nitrofuran drugs. This arose as a result of the finding of global misuse of these drugs in food animal production. The manufacturers validated the test kits in a range of matrices.

The European Commission has established Minimum Required Performance Limit for the nitrofuran drugs in poultry meat and aquaculture products (1.0 µg/kg) by Commission Decision 2003/181/EC. This was subsequently extended to cover all matrices tested by Commission Decision 2005/34/EC. The tests kits developed by FoodBRAND meet fully and indeed exceed the Minimum Required Performance Limit requirements established by these decisions.

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Queen's University Belfast
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