| Aflatoxins(AFs) are a kind of strong carcinogenic substances. The most poisonous derivatives of AFs is Aflatoxin B1(AFB1), which can cause liver lesions and lead to liver cancer. Feed and crops are easily contaminated by AFB1 because of high water content and unsuited storage temperature. AFB1 is difficult to be destroyed during the subsequent processing due to its high-temperature resistance. Therefore,AFB1 is very dangerous to humans. In this paper, colloidal gold immunochromatograohic assay(CG-ICA) and fluorescent microsphere immunochromatograohic assay(FM-ICA) were established to detect AFB1 quantitatively in feed and food. Based on these two methods, recovery experiment was carried out with spiked feed and food samples. This experiment is expected to provide data support for the subsequent production.Firstly, quantitative CG-ICA was established for the detection of AFB1 quantitatively. For CG-ICA, qualitative mode is different from quantitative mode. In the qualitative analysis, T-line signal is expected to disappear completely. But in the qualitative analysis T-line and C-line signal should be ensured in the linear range, and the T/C value should follow the change of the concentration. After a series of experiments, the results showed optimum condition: labeling pH is 6.0, the concentration of antigen on T-line is 1.5mg/mL, the concentration of antibody labeled on the colloidal gold is 4 μg/mL, and the dosage of gold-labeled antibody is 1.2 μL.The balance time of the test strip were optimized. The sensitivity, repeatability,stability and shelf life were also evaluated under the optimal conditions. Then the CG-ICA was used for quantitative detection of AFB1 in feed and food samples. The results suggested that the sensitivity of the assay was 5.7 ng/L. The sensitivity of the method has a great improvement compared to that of the qualitative detection strip judged by naked eyes. Since the assay can provide such a high sensitivity, a simple pretreatment method that dilute a lot of times after solvent extraction can be selected.This pretreatment method can not only reduce the substrate but also avoid complex experimental steps. For liquid samples such as soy sauce, they can be dilute directly for the test. Stability test proved that the method has a good stability, but newstandard curve in different batches is needed. The results of accelerated aging experiment indicated that the strip tended to be stable after 14 days when stored at room temperature and its shelf time was more than a year. A series of standard addition recovery efficiency of different substrates were obtained and several rules were summarized, providing supporting data for on-site detection.As a kind of label material, FM has been widely used in chromatography reaction because it has a lot of advantages such as good stability, cheapness, visible fluorescence and so on. This article chose fluorescent microspheres(FM) as a new label material to establish an immunochromatography method which is combined with fluorescent reader to detect AFB1 quantitatively. FM and monoclonal were combined into complexes based on EDC mediated. After experiments, several optimized conditions were obtained: antigen concentration on T-line is 1.5 mg/mL,second antibody concentration on C-line is 0.1 mg/mL, dosage of FM-AFB1 is 5 μL and the balance time of the strip was adjusted. FM-ICA was established under the optimal conditions. The results indicated that the sensitivity of the assay was 8.4 ng/L.Due to the high sensitivity of the method, simple dilution method for the pretreatment of soy sauce can be chosen. Four kinds of soy sauce were tested and the standard addition recovery rate and standard deviation were good enough for using. The results proved that the strip can be used in on-site and quantitative detection of AFB1. |
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