Search

The pre-aflatoxin protein biomarker PAB-01 is a key target for early predicting of aflatoxin occurrence risk. Improving its detection sensitivity is significant for the early evaluation of potential risks. In this study, we obtained the monoclonal antibody 6A6 against PAB-01 through hybridoma technology. A sandwich ELISA was developed using pairwise combinations of PAB-01 nanobody, monoclonal antibody, and polyclonal antibody. It was found that the nanobody as the capture antibody and monoclonal antibody as the detection antibody exhibited the highest sensitivity, with a detection limit of 0.92 ng/mL. Based on this, a rolling circle amplification-based immunoassay (RCA-EIA) was established, with a linear range of 0.056–41.152 ng/mL and a detection limit of 0.033 ng/mL. Recovery rates in spiked peanut and corn samples ranged from 94.49% to 127.50%. This method successfully identified the presence of the PAB-01 protein in maize samples 10 days prior to the aflatoxin production. Based on PAB-01 concentration classifications, the maize samples were categorized into different risk levels. This development is significant for early warning and risk stratification management of aflatoxin occurrence, providing a highly effective tool in ensuring food safety. • A new monoclonal antibody against PAB-01 protein was obtained. • VHH capture and mAb detection yielded the highest sensitivity in sandwich ELISA. • A rolling circle amplification (RCA)-based sandwich ELISA method was established. • RCA-EIA detected PAB-01 giving a 10-day early aflatoxin warning in corn. • Potential risk of aflatoxin occurrence was classified based on PAB-01 concentration. [ABSTRACT FROM AUTHOR]

Abstract: Mycotoxins are highly toxic contaminants and have induced health threat to human and animals. Aflatoxin B1 (AFB1), ochratoxin A (OTA) and zearalenone (ZEA) commonly occur in food and feed. A multi-component immunochromatographic assay (ICA) was developed for rapid and simultaneous determination of these three mycotoxins in agro-food. The strategy was performed based on the competitive immunoreactions between antibody-colloidal gold nanoparticle conjugate probes and mycotoxins or mycotixin antigens. Each monoclonal antibody specially recognize its corresponding mycotoxin and antigen, and there was no cross reactivity in the assay. Three mycotixin antigens were immobilized as three test lines in the nitrocellulose membrane reaction zone, which enable the simultaneous detection in one single test. The visible ICA results were obtained in 20min. The visual detection limits of this strip test for the AFB1, OTA and ZEA were 0.25ng/mL, 0.5ng/mL and 1ng/mL, respectively. The assay was evaluated using spiked and naturally contaminated peanuts, maize and rice samples. The results were in accordance with those obtained using enzyme-linked immunosorbent assay. In summary, this developed ICA could provide an effective and rapid approach for onsite detection of multi-mycotoxin in agro-food samples without any expensive instrument. [Copyright &y& Elsevier]

• A duplex phage-display immuno-polymerase chain reaction (PD-IPCR) was developed. • PD-IPCR was shown to be a sensitive method for mycotoxins detection. • The PD-IPCR was used successfully to evaluate aflatoxins and zearalenone in grains. An anti-idiotypic nanobody-phage display-mediated immuno-polymerase chain reaction (PD-IPCR) method was developed for simultaneous quantitative detection of total aflatoxins and zearalenone in cereals. Two phages, displaying the variable domain of the heavy chain anti-idiotypic nanobody that binds aflatoxin- or zearalenone-specific monoclonal antibody (1C11 or 2D3), were used as competitors for corresponding analytes. Specific DNA sequences encoding anti-idiotypic nanobodies were used to design the primers for PCR amplification. The results indicated that detection limits for total aflatoxins and zearalenone in a sample were 0.03 and 0.09 ng mL−1, respectively. Recoveries of spiked aflatoxins and zearalenone were 80–118% and 76.7–111%, respectively. Validation results were in good agreement with the gold-standard high-performance liquid chromatography method. This report is the first to describe PD-IPCR for simultaneous quantitative detection of total aflatoxins and zearalenone in cereals. [ABSTRACT FROM AUTHOR]