Development and validation of an indirect competitive enzyme-linked immunosorbent assay for monitoring quinoxaline-2-carboxylic acid in the edible tissues of animals

The feed drug additive carbadox is a suspected carcinogen and mutagen. To monitor effectively residues of carbadox in the edible tissues of food-producing animals, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect quinoxaline-2-carboxylic acid, the marker residue of carbadox, was developed. Several haptens were synthesised and conjugated to the carrier protein. Nine female New Zealand white rabbits were immunised with the immunising conjugates to produce polyclonal antibodies according to the designed schemes of immunisation. The highly specific antibody that was very sensitive to N-butylquinoxaline-2-carboxamide with an IC50 value of 7.75 mu g l(-1) was selected for the development of an ic-ELISA. The standard curves based on the N-butylquinoxaline-2-carboxamide matrix calibration ranged from 0.2 to 51.2 mu g l(-1). The decision limit and detection capability of the ic-ELISA were 0.60 and 0.83 mu g kg(-1) for liver and 0.68 and 0.79 mu g kg(-1) for muscle of swine, respectively. The recoveries were 57-108% with coefficients of variation of less than 20% when the quinoxaline-2-carboxylic acid was spiked into liver and muscle with the concentrations of 1.0-20.0 mu g kg(-1). Excellent correlations between the results of the ic-ELISA and an HPLC method (r = 0.9956 - 0.9969) were observed for incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for screening residues of carbadox in the edible tissues of food-producing animals.