Simultaneous determination for A. flavus-metabolizing mycotoxins by time-resolved fluorescent microbead or gold-enabling test strip in agricultural products based on monoclonal antibodies.

To simultaneously detect two metabolites of Aspergillus flavus, namely, cyclopiazonic acid (CPA) and aflatoxin (AFT), an ultrasensitive monoclonal antibody (mAb) YTT-2 against CPA was developed and characterized, with sensitivity to CPA of 1.32 ng mL-1. Along with the previously homemade mAb 1C11 against AFT, two mAbs were used to develop time-resolved fluorescence immunoprobes or gold immunoprobes. We developed two multiple-analyte paper immunosensors including time-resolved fluorescent immunochromatographic assay (TRFICA) and gold immunochromatographic assay (GICA) for the simultaneous determination of CPA and AFT. The TRFICA showed limits of determination (LODs) of 0.21 and 0.004 ng mL-1, while the GICA showed LODs of 0.33 and 0.01 ng mL-1 for CPA and AFT, respectively. To validate the specificity of the two rapid immunoassays, rice, corn and peanut samples were spiked with different concentrations of CPA and AFT. The two methods showed satisfactory recoveries (76.39~90.82% for CPA and 84.60~94.45% for AFT) and coefficients of variation of 3.50~7.80% for CPA and 4.12~13.90% for AFT. The results indicated that the TRFICA could complete the test within 5 min and had lower LODs and linear ranges, compared with that of GICA. The method developed in this work can be widely applied to the rapid and quantitative simultaneous determination of multiple harmful metabolites in fungi for food safety and health care. Graphical abstract.