Fusarium toxins, a group of mycotoxins, can be produced by Fusarium fungi under temperate climatic conditions on agricultural commodities, mainly cereals, in field as well as during storage. As a defensive response of the host plant, Fusarium toxins can be metabolized by forming modified mycotoxins, often called "masked" mycotoxins. It has been shown that many modified forms are hydrolysed into the parent mycotoxin during digestion. In order to protect consumer health from the risk of exposure to modified and parent forms of Fusarium toxins, the development of rapid, sensitive and reliable methods for their simultaneous determination in cereals is highly demanded. Currently, fluorescence polarization immunoassay (FPIA) is getting the attention as a screening tool in food safety control due to its simplicity, rapidity, cheapness and reliability. The focus of our work is to develop and validate quantitative FPIAs for simultaneous determination of DON and its acetylated (3-acetyl-DON, 15-acetyl-DON) and glycosylated forms (DON-3-glucoside) and T-2/HT-2 toxins and their glycosylated forms (T2-glucoside, HT2-glucoside) in wheat. A fluorescein-label (tracer) of DON (DON-FL) and four T2- and HT2-fluerescein tracers (T2-FL, HT2-FL1a, HT2-FL1b and HT2-FL2) were synthesized and purified. The assessment of the antibody-tracer binding was performed using four DON monoclonal antibodies (MAbs), and ten T2-glucoside MAbs, one HT-2 MAb and two T-2 MAbs, at different concentrations. Concerning the FPIA for the determination of DON, its acetylated and glycosylated forms, the highest antibody-tracer binding was observed for the clone 22/DON-FL combination, while in the FPIA for the determination of T-2/HT-2 toxins and their glycosylated forms, the highest bindings were observed for thirteen T2-glucoside MAbs with T2-FL and HT2-FL1b combinations, as well as for HT-2 MAb/HT2-FL1a combination and for T-2 MAb/T2-FL and HT2-FL1a combinations. Competitive FPIAs were performed with the selected antibody combinations. In particular, FPIA for the determination of DON, its acetylated and glycosylated forms showed IC50=23.5 ng/mL for DON and exhibited 204%, 45% and 11% as cross-reactivity, respectively, for 3-acetyl-DON, DON-3-glucoside and 15-acetyl-DON. While, among the selected combinations for T-2 and HT-2, the HT-2/HT2-FL1a combination exhibited 80% as cross-reactivity for T-2 and its glycosylated form and the highest sensitivity, with IC50 = 2.0, 2.6 and 2.7 ng/mL for HT2, T-2 and T2-glucoside, respectively. These findings showed the applicability of the developed FPIAs to the determination of parent and modified mycotoxins, expressed as sum, in solution. This work was supported by the MYCOKEY project which has received funding from the European Union's Horizon 2020 research and innovation programme under Grant Agreement No 678781.
Fluorescence polarization immunoassays for the determination of Fusarium toxins
Michelangelo Pascale;Marina Cortese;Vincenzo Lippolis
2017
Abstract
Fusarium toxins, a group of mycotoxins, can be produced by Fusarium fungi under temperate climatic conditions on agricultural commodities, mainly cereals, in field as well as during storage. As a defensive response of the host plant, Fusarium toxins can be metabolized by forming modified mycotoxins, often called "masked" mycotoxins. It has been shown that many modified forms are hydrolysed into the parent mycotoxin during digestion. In order to protect consumer health from the risk of exposure to modified and parent forms of Fusarium toxins, the development of rapid, sensitive and reliable methods for their simultaneous determination in cereals is highly demanded. Currently, fluorescence polarization immunoassay (FPIA) is getting the attention as a screening tool in food safety control due to its simplicity, rapidity, cheapness and reliability. The focus of our work is to develop and validate quantitative FPIAs for simultaneous determination of DON and its acetylated (3-acetyl-DON, 15-acetyl-DON) and glycosylated forms (DON-3-glucoside) and T-2/HT-2 toxins and their glycosylated forms (T2-glucoside, HT2-glucoside) in wheat. A fluorescein-label (tracer) of DON (DON-FL) and four T2- and HT2-fluerescein tracers (T2-FL, HT2-FL1a, HT2-FL1b and HT2-FL2) were synthesized and purified. The assessment of the antibody-tracer binding was performed using four DON monoclonal antibodies (MAbs), and ten T2-glucoside MAbs, one HT-2 MAb and two T-2 MAbs, at different concentrations. Concerning the FPIA for the determination of DON, its acetylated and glycosylated forms, the highest antibody-tracer binding was observed for the clone 22/DON-FL combination, while in the FPIA for the determination of T-2/HT-2 toxins and their glycosylated forms, the highest bindings were observed for thirteen T2-glucoside MAbs with T2-FL and HT2-FL1b combinations, as well as for HT-2 MAb/HT2-FL1a combination and for T-2 MAb/T2-FL and HT2-FL1a combinations. Competitive FPIAs were performed with the selected antibody combinations. In particular, FPIA for the determination of DON, its acetylated and glycosylated forms showed IC50=23.5 ng/mL for DON and exhibited 204%, 45% and 11% as cross-reactivity, respectively, for 3-acetyl-DON, DON-3-glucoside and 15-acetyl-DON. While, among the selected combinations for T-2 and HT-2, the HT-2/HT2-FL1a combination exhibited 80% as cross-reactivity for T-2 and its glycosylated form and the highest sensitivity, with IC50 = 2.0, 2.6 and 2.7 ng/mL for HT2, T-2 and T2-glucoside, respectively. These findings showed the applicability of the developed FPIAs to the determination of parent and modified mycotoxins, expressed as sum, in solution. This work was supported by the MYCOKEY project which has received funding from the European Union's Horizon 2020 research and innovation programme under Grant Agreement No 678781.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
