Development of enzyme-linked immunosorbent assay test kit for 1-aminohydantoin detection

Abstract

CPAHD, a carboxyphenyl derivative of AHD, was prepared and conjugated to immunogenic carrier protein and used in mice immunization. After conventional somatic cell fusion technique, a monoclonal antibody (MAb), clone number 28/10E/8B/1E, was obtained. The antibody was found to be highly specific for nitrophenylbenzal-1-aminohydantoin (NPAHD) and exhibited high sensitivity with the 50% inhibition concentration (IC50) at 41.8 part per billion (ppb). This antibody showed specificity only against NPAHD, CPAHD and nitrofurantoin, and no cross reactivity to unrelated drugs was found. Using the partially purified antibody, the antibody captured competitive indirect ELISA format (biotinylated antibody) exhibited the best sensitivity with IC50 and limit of detection (LOD) values of 4.905 and 0.113 ppb. When applied ELISA to detect AHD in shrimp samples, the recovery rate are in the range of 85% – 118% for intra-assay and 98% – 111% for inter-assay. Analysis of AHD in fortified shrimp samples using the developed ELISA and LC-MS/MS was comparable. Detection capability (CCβ) of the developed ELISA was found to be 0.40 ppb.