Development and validation of an enzyme-linked immunosorbent assay for rapid detection of multi-residues of five quinoxaline-1,4-dioxides in animal feeds

In this study, an enzyme-linked immunosorbent assay (ELISA) was developed for the simultaneous quantitative determination of five quinoxaline-1,4-dioxides (olaquindox, carbadox, mequindox, quinocetone and cyadox) in porcine and chicken feeds. Polyclonal antibodies (PAbs) with group-specificity to quinoxaline-1,4-dioxides were raised in rabbits after immunization with quinoaxline-2-carboxylic acid-para-aminobenzoic-bovine serum albumin conjugate (QCA-PABA-BSA). The specificity of anti-sera was determined by competitive indirect ELISA (ciELISA), and the 50% inhibitions (IC₅₀) of quinocetone (QCT), cyadox (CYA), carbadox (CBX), mequindox (MEQ) and olaquindox (OLA) were 8.6, 13.1, 17.3, 24.5 and 26.4 ng ml⁻¹, respectively. The limit of detection (LOD) for the QCT, CYA, CBX, MEQ and OLA were as low as 0.41, 0.59, 0.67, 0.80 and 0.86 ng g⁻¹, respectively. On the basis of PAb's strong cross-reactivity to QCT (100%), CYA (49.9%), CBX (66.2%), MEQ (35.3%) and OLA (32.8%), no cross-reactivity was found with other antibiotics, indicating that the assay displayed not only high-sensitivity but also high-specificity. When used to analyse animal feed samples, acceptable recovery rates of 71.6–92.9% and coefficients of variation of 4.1–10.8% were obtained. The ciELISA for 10 spiked samples was confirmed by high-performance liquid chromatography (HPLC) with a high correlation coefficient of 0.9959 (n=10). Therefore, the optimised ciELISA may be a potential analytical tool for monitoring quinoxaline-1,4-dioxide residues in animal feed.