| Mycotoxins are a kind of small molecular secondary metabolites produced by fungi,more than 75%of which are mixed contamination of mycotoxins in agricultural products,Zearalenone(ZEN)and Ochratoxin A(OTA)are two common mycotoxins that contaminate corn samples at the same time.ZEN is mainly an estrogenic metabolite secreted by Fusarium graminearum in the absence of nutrients,which affects the reproductive system of animals,can cause the increase of estrogen in livestock and significant economic losses to animal husbandry.OTA is a toxic secondary metabolite produced by molds.It is highly toxic and widely distributed.It damages the liver and kidney,destroys the immune system,and has carcinogenic,teratogenic and mutagenic effects.Because of its unique quantum size effect,poly-chromatic quantum dots(QDs)with different emission wavelengths can be obtained by changing the particle size of quantum dots.When excited by a single light source,the fluorescence emitted by different quantum dots is different,which has great application potential in the field of multiple monitoring of mycotoxins.In this study,Zearalenone complete antigens ZEN-BSA and ZEN-OVA,were synthesized by oximation method and carbodiimide method,and identified by UV scanning spectrum and polyacrylamide gel electrophoresis(SDS-PAGE).The results showed that the artificial antigen was successfully prepared.Through immunological identification,ZEN-BSA was used as immunogen to immunize Balb/c mice,and the tail vein serum of mice was collected one week after three immunization.The titer and half inhibitory concentration(IC50)of mouse polyantiserum were determined by using ZEN-OVA as coating antigen.The titer of mouse No.2 was1:2.048×105 and the IC50 was 1.73 ng/m L.The hybridoma cell line 4D7 which can stably secrete anti-ZEN monoclonal antibody(m Ab)was obtained by cell fusion technique.The anti-ZEN monoclonal antibody was obtained by in vivo induction method and caprylic acid-ammonium sulfate precipitation method.The titer of the monoclonal antibody was 1:5.12×105,IC50 was 0.16 ng/ml and the affinity constant was 2.23×109 L/mol.There was no cross reaction with other mycotoxins.The hybridoma cell line 2G3,which can secrete anti-OTA monoclonal antibody,was obtained by the same method.The titer of anti-OTA monoclonal antibody was1:2.56×105,IC50 was 7.18 ng/ml,affinity constant was 1.74×109 L/mol,and the cross-reaction rates with Ochratoxin B(OTB),Ochratoxin C(OTC)and Ochratoxin D(OTD)were 8.76%,0.83%and 0.79%,respectively,and less than 0.01% for other mycotoxins.Two kinds of immunofluorescence probes,anti-ZEN-m Ab-QDs and anti-OTA-m Ab-QDs,were prepared by coupling green quantum dots with anti-ZEN monoclonal antibody and orange-red quantum dots with anti-OTA monoclonal antibody by the carbodiimide(EDC)method.ZEN-OVA and OTA-OVA were simultaneously coated in a single well of black microplates with high binding capacity,and a multiple fluorescence labeled immunosorbent assay(M-FLISA)for simultaneous quantitative detection of two mycotoxins was established.According to the M-FLISA,the IC50 of ZEN was 0.034 ng/m L,the limit of detection(LOD)in the maize was 4.32×10-5 ng/m L and the added recovery rate was 93.15%-101.90%.The IC50 of OTA was 1.17 ng/m L,the LOD was 0.018 ng/m L,the added recovery rate was 95.29%-102.43%,and the coefficient of variation(CV)was less than 9%.The accuracy and reliability of the method were verified by high performance liquid chromatography(HPLC).The results showed that there was a excellent consistency,which proved that multiple fluorescence labeled immunosorbent assay was suitable for the simultaneous quantitative detection of many kinds of mycotoxins. |
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