Preparation Of Immunochromatographic Strip Test For Simultaneous Detection Of Aflatoxin B₁ And Zearalenone

Aflatoxin B1 (AFB1) and zearalenone (ZEA) are harmful substance existed in cereals and secondary metabolites produced by fungus which contaminated cereals. AFB1 is a highly toxic and carcinogenic substance, which can induce a variety of cancers; ZEA has functions as female hormone and may destroy the normal reproductive development of human and animal. AFB1 and ZEA commonly exists in cereals, serious threat to health of human.Actual methods are time consuming and need professional equipment, thus it is very important to develop Immunochromatographic strip test for fast, convenience and accurate simultaneous detection of Aflatoxin B1 and Zearalenone.The main results of our research are as follows:1. The conjugation of AFB1/ZEA and ovalbumine (OVA) was produced according to the method of DCC catalyze. The protein concentration of AFB1-OVA and ZEA-OVA were 1.68 and 2.25 mg/mL, respectively; They emerged a new absorption peak at 360nm and 310nm by UV-Spectra scanning, respectively; Through SDS-PAGE analysis, AFB1-OVA has a good purity, there were little impurity in ZEA-OVA; Molar ratio of two conjugations were 12.3 mole AFB1 and 16.5 mole ZEA to OVA, respectively. Conjugations have good stability and can keep stable results in 300 days stored at -20℃.2. Colloidal gold particles (diameter 40nm) were prepared through reducing HAuCl4·3H2O by sodium citrate, solution was characterized by UV-Spectra and transmission electron microscope (TEM), particles were uniform , the average diameter of particles were 40.2±0.4nm; Anti-AFB1 monoclonal antibody was prepared by serum-free culture technique and using affinity chromatography purify it. The titer of anti-AFB1 monoclonal antibody and anti-AFB1 monoclonal antibody were 1:8×104 and 1:1.28×105, respectively. Through SDS-PAGE analysis, affinity chromatography can remove impurity protein in supernatant fluid. Two antibodies all have a good purity. The optimal pH for both colloidal gold particles conjugated with AFB1 and ZEA McAb were 7.0 and the optimal protein concentration were 5.0 and 2.5μg/mL, respectively. AFB1-OVA and ZEA-OVA were coated on the Milipore 135s nitrocellulose membrane and optimal concentration was 1.0 and 2.0 mg/mL, respectively. Based on above prepared strip test which can simultaneous qualitative detected AFB1 and ZEA in sample and the limit of detection were 5 and 50μg/kg.3. The optimal methanol content of sample extracting solution for AFB1 strip test,ZEA strip test and simultaneous detected AFB1 and ZEA strip test were 30%,20% and 20%, respectively.There was no cross reaction to Aflatoxin M1 and deoxynivalenol. The expiration date of three strip test were 1 year,1 year and 9 months. 92 corn samples from northern areas were analyzed by strip test,ELISA and TLC, 5 samples have higher level than 5.0ng/mL for AFB1 and 9 samples have higher level than 5.0ng/mL for ZEA. Results of the strip test were in good agreement with those obtained by ELISA and TLC.