Full
Outline
To establish a method for the rapid detection of 3 kinds of common and highly toxic mycotoxins in agricultural products—aflatoxin B1 (AFB1), T-2 toxin and zearalenone (ZEA) by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).
High-affinity monoclonal antibodies were used as recognition elements, based on the ic-ELISA principle, and key ic-ELISA parameters—including coating antigen type and concentration, antibody dilution ratio, antigen diluent composition and competition incubation time—were systematically optimized. Independent detection systems for AFB1, T-2 toxin and ZEA were established and their performance was validated through spiked recovery experiments.
The complete antigens for the 3 kinds of mycotoxins were successfully conjugated. The titers of the corresponding monoclonal antibodies were 2.56×104 for AFB1, 8.19×105 for T-2 toxin and 2.56×104 for ZEA, with no cross-reactivity observed with other mycotoxins.The optimal detection conditions were as follows: For AFB1—coating antigen AFB1-bovine serum albumin(BSA) at 8 μg/mL, primary antibody dilution of 1:6400, antigen diluent comprising 0.010 mol/L phosphate-buffered saline (PBS) (pH 9.0) containing 10% methanol and 0.010% Tween-20 and a competition incubation time of 45 min;for T-2 toxin—coating antigen T-2-BSA at 8 μg/mL, primary antibody dilution of 1:25600, antigen diluent comprising 0.005 mol/L PBS (pH 7.4) containing 10% methanol and a competition incubation time of 30 min; for ZEA—coating antigen ZEA-ovalbumin (OVA) at 0.5 μg/mL, primary antibody dilution of 1:1600, antigen diluent comprising 0.010 mol/L PBS (pH 6.0) containing 10% methanol and a competition incubation time of 60 min. The method performance parameters were as follows: Limits of detection of 0.0273 ng/mL (AFB1), 0.183 ng/mL (T-2 toxin)and 1.09 ng/mL (ZEA), with corresponding linear ranges of 0.047–0.588, 0.288–7.493 and 2.319–69.533 ng/mL. Spiked recoveries were 104.0%–110.0%, 107.3%–108.3% and 97.9%–112.3%, respectively and all coefficient of variations were below 15%.
The established ic-ELISA method demonstrates high sensitivity, good reproducibility and strong specificity, enabling the rapid screening of AFB1, T-2 toxin and ZEA in grain samples. It provides reliable technical support for the monitoring and risk assessment of mycotoxin contamination in agricultural products.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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