Study On Kapld Immunology Screening Methods Of Fumonisin B₁and Deoxynivalenol In Corn

Fumonisins are water-soluble toxic metabolites of a family of mycotoxins produced by Fusarium moniliforme and natural contaminants of corn and corn products all over the world. Among these fumonisin derivatives, FB1is the commonest one and also the main cause of the toxic effect of FBs. FB1is neurotoxic, immunotoxic and carcinogenic. Deoxynivalenol, also known as vomitoxin, is a series of toxic analogue trichothecenes, which are the secondary fungal metabolites being produced predominantly by F.graminearum and F. culmorum. DON is a common mycotoxin contaminants of wheat, barley, maize, and cereal-based food products. DON is immunotoxic, embryo toxic and cytotoxic. Now the main methods to determin FB1and DON are biological assay, physical and chemical detection and immunoassay. Immunoassay is suitable for large quantity of samples for rapid quantitating detection on site due to its high specificity, sensitivity, simple sample preparation. In this study, a direct competitive Enzyme linked immunoassay (dc-ELISA) and Polyvinylidene fluoride (PVDF) membrane-based direct competition immunoassay were developed to detect FB1, DON and simultaneous detect FB1and DON in corn samples, respectively.FB1was activated by1-ethyl-3-(3-dimethy-lamiopropyl)carbodiimide hydrochloride (EDC), and conjugated to ovalbumin (OVA) as the FB1detecting antigen (FB1-OVA). DON was activated by N,N-carbonyldiimidazole (CDI), and conjugated to bovine serum albumin (BSA) as the DON detecting antigen (DON-BSA). Horseradish peroxidase (HRP) was successfully coupled to anti-FB1monoclonal antibody (McAb) and anti-DON McAb at different mass ratio by cyanuric chloride method (CC), periodate method (PI) and glutaraldehyde method (GA). The Dc-ELISA results showed that titer of anti-FB1McAb-HRP produced by cyanuric chloride fast method was the highest, while IC50was the lowest. McAb and HRP mass ratio of CC-1-FB1McAb-HRP was1:2, the molar ratio was2.92, the enzyme rate was51%and labeling rate was0.84454; IC50of anti-DON McAb-HRP produced by glutaraldehyde method was the lowest, repeatable was best, McAb and HRP mass ratio of GA-2-DON McAb-HRP was1:20, the molar ratio was2.69, the enzyme rate was3.345%and labeling rate was0.6935.A Dc-ELISA method for detecting FB1in corn samples depending on the CC-l-FB1McAb-HRP was established. According to checkerboard titration, the optial working concentration of FB1-OVA was1μg/mL and CC-1-FB1McAb-HRP was diluted to1/32000, IC50was2.97ng/mL, and the linear working concentration range was0.56～15.6ng/mL. Both intra-assay coefficient of variation and inter-assay coefficient of variation were lower than10%. The recovery rate was between83.3and93.2%. The cross reactivity of CC-1-FB1McAb-HRP to FB2was16%, and no cross reaction was found with other common mycotoxins.A Dc-ELISA method for detecting DON in corn samples depending on the GA-2-DON McAb-HRP was established. According to checkerboard titration, the optial working concentration of DON-BSA was1μg/mL and GA-2-DON McAb-HRP was diluted to1/16000, IC50was77.267ng/mL, the linear working range was16.61～359.36ng/mL. Intra-assay coefficient of variation was lower than10%and inter-assay coefficient of variation was16.66%. The recovery rate was between90.28and97.7%. The cross reactivity of GA-2-DON McAb-HRP no cross reaction was found with other common mycotoxins.PVDF membrane-based direct competition immunoassay for simultaneous detection of FB1and DON in corn samples was established. The whole process only has one step, and a batch of extracted samples can be analyzed within15mins. The detection limits for FB1and DON were2.5ng mL-1and50ng mL-1, respectively. The advantages were good stability, period of validity was not less than180days, the result can be determined accurately and easily, economical and practical. It is the rapid method for the screening analysis in samples on-site test.The concentration of FB1and DON were determined in35corn samples by the PVDF membrane-based immunoassay and Dc-ELISA, the results showed no significant difference with the ELISA kit and HPLC.