Development Of Colloidal Gold Immunochromatographic Assay For Rapid Detection Of Aflatoxin B1

Aflatoxin B1 are carcinogenic secondary metabolites produced primarily by Aspergillus flavus and A. parasiticus. They can be present in grains, nuts, cottonseed and other commodities associated with human food or animal feeds. Aflatoxin B1 has been implicated in human health and animal feeds. So, most controlling government agencies worldwide have regulations regarding the amount of aflatoxin B1 allowed in human and animal foodstuffs, and detected it as compellent standard. The detection methods of AFB1 now are complicated to operate and need special instruments, which cannot meet the requirement of rapid detection on the spot. Therefore, development of a convenient and rapid method for AFB1 detection is extremely important and necessary.In this study, dot immunogold assay (DIGA) based on monoclonal antibody (McAb) and immunogold labeling technique (ILT) was developed for the AFBI detection according to competitive inhibition principle.Adopting vivo culture method, hyridoma cell 2A5 capable of secreting McAb against AFBI continually, were injected into BLAB/c mice to produce ascites. The ascites of the mice was collected and purificated by caprylicacid-ammonium sulfate precipitation (CA-AS) to obtain the monoclonal antibody. The McAb was determined with coating antigen and the results showed that the concentration of the McAb was 4mg/mL, the titer of the McAb was 1:6.4×104 and the 50% inhibition concentration was 0.218 ng/mL. The specificity of the McAb was well.Trisodium citrate with aqueous gold chloride were warmed up and mixed to make colloidal gold. Colloidal gold immunochromatographic assay(GICA)was developed for the detection of AFB1 based on competitive inhibition principle. In this system, McAb against AFB1 conjugated to colloidal gold served as the analysis probe, complete antigen AFB1-BSA acted as the coating antigen(test line), goat anti-mouse IgG was immobilized on the membrane acting as the control dot(control line). The optimal diameter of colloidal gold particles was about 24 nm. The optimal coating antigen was 0.3 mg/ml, the colloidal gold labeled antibodies was 0.5μg/mL.The GICA sensitivity for AFB1 detection was 3 ng/mL by visual observation. The detection time was about 5 to 10 min. This method had good specificity for AFB2,AFM1,Ochratoxin B and Zearalenone, and obvious cross-reactivity with AFG1 and AFG2. Polluted samples were prepared by adding six different concentrations of AFB1(1 ng/mL,2ng/mL,3 ng/mL,5 ng/mL,10 ng/mL,20 ng/mL) to uncontaminated corn flour. The results were analysed by DIGA and ELISA, respectively. The concentration of AFB1 less than 2 ng/mL, there were two red lines on the nitrocellulose membrane. The concentration of AFB1 more than 3 ng/mL, only control line was red. And ELISA as the parallel experiment, two results were correspond.The major advantages of the strip were that results could be obtained within 10 minutes and that all needed reagents were included in the strip. The strip could be used to detect the AFB1 of rapid detection, not need any special instruments.