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

- AFB2 and AFG2 as competitors for screening broad-specific monoclonal antibodies against aflatoxins.
- A new monoclonal antibody 5H3 with uniform affinity towards the major aflatoxins was prepared.
- Magnetic beads as immobile phase improved the sensitivity of ELISA.
- A reliable magnetic beads-ELISA for the detection of total aflatoxins was developed.
- The detection of the magnetic beads-ELISA for the total aflatoxins was 0.21 ng/g in maize.

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, AFB1+AFB2+AFG1+AFG2) 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 AFB1, 0.04 ng/mL for AFB2, 0.05 ng/mL for AFG1, 0.06 ng/mL for AFG2. 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 ng/g 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.