Study On Gold Labeled Immunochromatographic Assay For Detection Of Deoxynivalenol In Foods

Deoxynivalenol(DON), as one fungi toxin belonging to the group of trichothecenes, is widely distributed in cereal and feedstuffs, and its rapid detection is important for food safety. However most of the currently used methods are time-consuming and also need expensive instruments, which cannot satisfy the requirement for a rapid, convenient, and on-site detection of the presence of DON in food samples.Thus,an immunochromatographic strip system using colloidal gold-labeled polyclonal antibody(PcAb) specific to DON as the maker was developed for rapid detection of DON in foods.The full-antigen of DON was first produced by conjugating DON to bovine serum albumin (BSA) according to the method of DCC. A molar ration of 9.7:1 between DON and BSA was abtained in the conjugation reaction from an initial molar ratio of 50:1.After the successful conjugation of BSA to DON was verified by ELISA and IR spectra, the PcAb to DON-BSA was then produced by immunizing rabbits. The results of characterizing PcAb showed a highest titer of 12800 with an IC50 of 10μg/mL, and its cross reactivity with T-2 toxin and NIV was 9.1% and 4.9% respectively.Colloidal gold were prepared through reducing HAuCl4·3H2O by sodium citrate, and it was then characterized by UV-spectra. The optimum pH for antibody labelling with colloidal gold (also called gold labeled probe) was 9.0, and the concentration of antibody was 0.066mg/mL,0.072 mg/mL. The probe colloidal gold particles, and antibody were analyzed by UV-spectra, and the results showed that the conjugation was successful.Combination of PcAb with colloidal gold particles was also characterized by IR spectra and fluorescence spectroscopy. The experimental resluts showed that the structure of PcAb was not changed, but its fluorescence can be quenched by the colloidal gold, and its power was strengthened as the concentration increased. However no changes of the emission spectrum profile was found .A rapid, simple and sensitive colloidal gold labeled immunochromatographic test(GICA) for detection of DON in foods was established. The optimum concentration of envelope antigen was 10μg/mL. A buffer system composed of PBS (pH 7.4)+10%methanol+0.05%Tween20 was used to detect the DON. The optimum particle diameter of colloidal gold was found to be 20nm, and the detection process could be finished within 15min, which is reproducible and stable. With visual observation, the lowest limit was found to be 1mg/L corresponding to 1mg/Kg DON in samples. The shelf-life of immunostrips was three months at room temperature and six months at 4℃.Comparing with commercial ELISA kit using naturally contaminated food samples, consistent result was obtained using the developed GICA method.