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This is the first report of QDs-FLISA to detect AHD developed based on a specific monoclonal antibody.The FLISA offers higher sensitivity in comparison with ic-ELISA.Excellent correlations of the ic-ELISA/LC–MS/MS and FLISA/LC–MS/MS data were observed for processed samples.Monitoring and rapid evaluation of nitrofurantoin metabolite, 1-aminohydantoin (AHD), are important for food safety and human health. Herein, we established the monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and quantum dots (QDs)—fabricated fluorescence-linked immunosorbent assay (FLISA). Monoclonal antibody specific to nitrophenyl derivative of AHD was derived from hybridoma cell lines 3.2.4/5A8. For another, CdTe core QDs with emission wavelength of 605 nm were also synthesized. The performances of the proposed ic-ELISA and FLISA were further examined and the corresponding results were also validated by standard LC–MS/MS analysis. The obtained results indicated that both ic-ELISA and FLISA exhibited good dynamic linear detection for NPAHD over the range from 0.1 to 3.0 ng mL−1. Meanwhile, proposed immunosorbent assays are characterized by satisfactory recovery rates of 81.5–113.7%. The experimental data suggested these two immunoassays could be facile, cost-effective and rapid tools for the prospective quantitative method for AHD analysis in food matrix.