Development of a new broad-specific monoclonal antibody with uniform affinity for aflatoxins and magnetic beads-based enzymatic immunoassay

To reduce the incidence of false-positive and false-negative results caused by high or low cross-reactivity (CR%) values of the antibodies for total aflatoxins (AFs, AFB(1)-FAFB(2)+AFG(1)+-AFG(2)) detection, a new broad specific monoclonal antibody (MAb) with uniform affinity, named 5H3, was developed. Moreover, magnetic beads (MBs) replaced microplates as immobile phase to improve the sensitivity of the enzymatic immunoassay. Then, a direct competitive enzyme-linked immunosorbent assay (ELISA) based on MBs (MBs-dcELISA) that could simultaneously detect the total AFs with similar sensitivity was developed. Following optimization of conditions, the half maximal inhibitory concentrations (IC50) of the MBs-dcELISA in buffer were 0.05 ng/mL for AFB(1), 0.04 ng/mL for AFB(2), 0.05 ng/mL for AFG(1), 0.06 ng/mL for AFG(2). The corresponding CR% values were 100%, 125%, 100% and 83.3%, respectively. The limit of detection (LOD) of the MBs-dcELISA for the total AFs was 0.21 ng/g with a working range from 0.22 ngig to 19.8 ng/g, and the recoveries for the total AFs ranged from 74.5% to 96.5% with coefficients of variation (CV) under 12.1% in spiked maize samples. In addition, the MBs-dcELISA was more sensitive than the conventional dcELISA. Finally, the MBs-dcELISA was applied to screen 9 naturally contaminated maize samples and 6 spiked samples and the results indicated a good agreement with that obtain by HPLC-MS/MS method. (C) 2017 Published by Elsevier Ltd.