Quantitative Determination of Aflatoxin B1 in Maize and Feed by ELISA and Time-Resolved Fluorescent Immunoassay Based on Monoclonal Antibodies

In this study, a highly sensitive monoclonal antibody (mAb) was developed for the detection of aflatoxin B-1 (AFB(1)) in maize and feed. Additionally, indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and time-resolved fluorescence immunoassay assay (TRFICA) were established. Firstly, the hapten AFB(1)-CMO was synthesized and conjugated with carrier proteins to prepare the immunogen for mouse immunization. Subsequently, mAb was generated using the classical hybridoma technique. The lowest half-maximal inhibitory concentration (IC50) of ic-ELISA was 38.6 ng/kg with a linear range of 6.25-100 ng/kg. The limits of detections (LODs) were 6.58 ng/kg and 5.54 ng/kg in maize and feed, respectively, with the recoveries ranging from 72% to 94%. The TRFICA was developed with a significantly reduced detection time of only 21 min, from sample processing to reading. Additionally, the limits of detection (LODs) for maize and feed were determined to be 62.7 ng/kg and 121 ng/kg, respectively. The linear ranges were 100-4000 ng/kg, with the recoveries ranging from 90% to 98%. In conclusion, the development of AFB(1) mAb and the establishment of ic-ELISA for high-throughput sample detection, as well as TRFICA for rapid detection presented robust tools for versatile AFB(1) detection in different scenarios.