Development Of Colloidal Gold Immuno Chromatographic Strip For Zearalenone

Zearalenone(ZEN)is a secondary metabolite mainly produced by Fusarium spp.It widely exists in different cereals,such as corn,barley,oat,wheat,rice and sorghum.It not only affects the economical and nutritional value of crops,but has certain toxic for human.At present,the analysis method used for determination of ZEN mainly include chromatography test and immunoassays.Immunoassays have higher sensitivity,strong specificity,lower cost,wider range of application.Colloidal gold immunochromatographic strip in the test could detect ZEN rapidly.In this experiment,on the basis of hybridoma cell line called 3D 10,which could produce monoclonal antibodies against ZEN stably,a Colloidal gold immunochromatographic strip for rapid detection of ZEN is developed.There are three parts in this paper.The first part:preparation of coated antigen and monoclonal antibodies.Zearalenone and carboxy-methoxylamine hemihy-drochloride were reacted to synthetize the hapten.The hapten was conjugated to ovalbumin(OVA)to form the complete antigens with NHS ester method.The hapten was identified by thin layer chromatography(TLC)and Electrospray ionization and mass spectrometry(ESI-MS).The complete antigen was identified by ultraviolet spectrum.The titre of the ascites produced by 3D10 reached 1×106,and the concentration of antibody was 3.8 mg/mL.The second part is the preparation of colloidal gold immunochromatographic strip.First of all,colloidal gold particles(diameter 30 nm)were prepared through reducing HAuCl4·3H2O by sodium citrate.Solution was characterized by visual observation,UV-Spectra and transmission electron microscope(TEM),and particles were uniform.Through the method of the naked eye observation,the optimal pH for colloidal gold particles conjugated with ZEN McAb were 7.5,and the optimal protein concentration was 9.6 ?g.ZEN-OVA and IgG were coated on the millipore 135 nitrocellulose membrane and optimal concentration for both were 1 mg/mL.The third part is the evaluation and application of the colloidal gold immunochromatographic strip.A range of indicators,including the specificity and the minimum detection limit,were evaluated.There was no cross-reaction to other mycotoxin such as Deoxynivalenol(DON),HT-2.T-2 and Aflatoxin B1(FB1),but showed high cross-reaction to the four kinds of analogues,such as a-zearalenol(a-ZOL),?-zearalenol(?-ZOL),a-zearalanol(a-ZAL),?-zearalanol(p-ZAL).The minimum detection limit of the colloidal gold strip was 30?g/kg.The strip was valid for two days under thirty-seven degrees celsius,but more than one week under four degrees celsius.10 samples were analyzed by colloidal gold immunochromatographic strip and HPLC-MS,7 samples have higher level than 30 ?g/kg.Results of the colloidal gold strip test were in good agreement with HPLC-MS/MS.