Study On Immunoassays For Simultaneous Detection Of Thiabendazole And Ochratoxin A

Thiabendazole,a systemic fungicide of benzimidazoles,commonly used for prevention of storage diseases like blue mold of citrus and growing diseases such as wheat scab and grape anthracnose caused by penicilium,aspergillus and gibberellic.Ochratoxin is a secondary metabolite of aspergillus and penicillium fungi,with strong hepatotoxicity,nephrotoxicity and carcinogenic effect.Improper use of pesticides in the control of fungal diseases will result in mixed contamination of pesticides and mycotoxins,which will seriously harm human safety and ecological environment.Therefore,it is necessary to establish simultaneous detection methods for effective monitoring.In this study,enzyme-linked immunosorbent assay(ELISA)for ochratoxin A(OTA)and two fluorescence immunoassay methods for simultaneous detection of thiabendazole and OTA were established by using monoclonal antibodies against thiabendazole and OTA,and were applied to the detection of thiabendazole and ochratoxin A in agricultural products.The main results were as follows:1.The artificial antigens of OTA were prepared by the active ester method,and the binding ratio of immunogen and coating antigen was 9:1 and 7:1,respectively.BALB/c female mice were immunized with immunogen by routine intraperitoneal injection.After cell fusion and cell screening,two monoclonal cell lines 6C3 and 3B1 with stable OTA antibody secretion were obtained,and the antibodies were prepared and purified.The sensitivity was preliminarily evaluated by indirect competitive enzyme-linked immunosorbent assay(ic-ELISA).Antibody 3B1,with the better sensitivity,was selected for the establishment of the immunoassays.2.ELISA was established based on the prepared OTA monoclonal antibodies.By optimizing the working concentration of antigen and antibody,and buffer solution(content of methanol,ionic strength and pH),the standard curve of this method under the optimal conditions was established,with the half inhibitory concentration(IC50)of 4.05 ng/mL and linear range of 0.92-20.99 ng/mL.The method had no cross reaction with other mycotoxins,showing it is of high specificity.The average recoveries of brown rice,wheat flour,corn and grape were 72.5-87.7%and the relative standard deviations(RSDs)were 1.7-5.3%.The method was verified by high performance liquid chromatography(HPLC),the correlation indicated that this method is accurate and reliable and can be applied to the detection of OTA residue in the agricultural samples.3.Lanthanide europium(Eu3+)was used as the fluorescence marker to label goat anti-mouse IgG antibody,and a high-sensitivity time-resolved fluorescence immunoassay(TRFIA)based on thiabendazole and OTA monoclonal antibody was established.TRFIA standard curves of thiabendazole and OTA were established under the optimal conditions(Eu3+-labled antibody with a dilution of 5000-fold,5%of methanol content,sodium ion concentration of 0.15 mol/L and pH value of 7.8).The IC50 and the limit of detection(LOD)for thiabendazole were 1.56 ng/mL and 0.21 ng/mL,while 1.03 ng/mL and 0.18 ng/mL for OTA,respectively.The sensitivity of both in the TRFIA was much higher than that in ELISA.The established TRFIA showed no significant cross-reaction with analogues of thiabendazole and other common mycotoxins.The average recoveries and the RSDs for thiabendazole in brown rice,wheat flour,corn and grape were in the range of 77.2%to 94.3%,and 0.9%to 5.6%,respectively,while 79.5%to 91.9%and 1.5%to 7.4%for OTA,respectively.The test results of real samples showed that the results of TRFIA were highly correlated with HPLC(R2=0.9984).The study showed that the established TRFIA can realize simultaneous detection of thiabendazole and OTA residues in agricultural products with high sensitivity and high efficiency.4.Based on magnetic separation,an upconversion fluorescence immunoassay method for simultaneous detection of thiabendazole and OTA was established.The artificial antigen of thiabendazole and OTA were coupled to magnetic nanoparticles(MNPs),respectively.The thiabendazole and OTA were conjugated to animo-functionalized upconversion nanoparticles(UCNPs),respectively.Under the effect of external magnetic field,MNPs was captured to achieve the purpose of separation and enrichment,and the concentration of analytes was negatively correlated with fluorescence intensity.The standard curves were established under the optimal conditions.The IC50 and the linear range for thiabendazole were 9.51 ng/mL and 0.36-94.17 ng/mL respecticely,while 4.40 ng/mL and 0.85-39.77 ng/mL for OTA.The method had no obvious cross reaction with structure analogues of thiabendazole and other mycotoxins.The average spiked recoveries of brown rice,wheat flour,corn and grape samples were 76.2-92.5%and 80.1-95.9%for thiabendazole and OTA,respectively.The RSDs were 1.0-6.5%and 1.2-6.6%for thiabendazole and OTA.The test results of real samples showed that the method was highly correlated with HPLC,which proved the method can apply to simultaneous detection of thiabendazole and OTA.