Preparation of high-affinity and high-specificity monoclonal antibody and development of ultra-sensitive immunoassay for the detection of 1-aminohydantoin, the metabolite of nitrofurantoin

1-Aminohydantoin (AHD), the residual marker of nitrofurantoin, is usually detected after derivatisation using the derivatisation reagent 2-nitrobenzaldehyde. Avoiding the antibody recognition of the derivatisation reagent is essential for the accurate detection of AHD residues. In this paper, a novel hapten called hapten D was designed, and then, a monoclonal antibody that did not recognise 2-nitrobenzaldehyde was prepared based on this novel hapten. An ultra-sensitive indirect competitive enzyme linked–immunosorbent assay (icELISA) was established under optimal conditions. The 50% inhibition concentration and limit of detection of AHD were 0.056 and 0.0060 ng/mL, respectively, which improved the sensitivity by 9–37-fold compared with the previously reported icELISA methods. The average recovery rates were 88.1%–97.3%, and the coefficient of variation was <8.6%. The accuracy and reliability of the icELISA were verified using liquid chromatography–tandem mass spectrometry. These results demonstrated that the developed icELISA is a useful and reliable tool.