Development Of Immumnoassay For Aflatoxin B₁

AFB1 is a toxic compound produced by traditional aflatoxin species of Aspergillus moulds. It is highly cytotoxic to mammalian cells, which has been shown recently that the compound is linked to an increased risk of cancer. It is significant to develop a rapid, high throughput screening methods for determination this compound. Immunochemical analysis demonstrated as simple, rapid, and cost-effective, high throughput alternative has been successfully developed as semiquantitative or quantitative screening tools in food safty. The major research contents and the results as follows:(1) Indirect AFB1-ELISA was established, and get the AFB1-ELISA assay kit. The IC50 of icELISA was 1.07μg/L, the limit of detection was 0.05μg/L, detection range of 0.15-4.05μg/L, the linear regression equation of y = -17.49x +109.03(R2 = 0.9945), the within-run and between-run CVs were 4.88% and 6.96% repectively, which showed the good stability of the icELISA. The recoveries of all kinds of sample were range from 80% to 110%. The accelerated testing method showed that the validity period could be over 1 year when assay kit were storaged in 4℃.(2) A direct competitive TRFIA was established which was more sensitive comparing with ELISA. Results showed the limit of detection was 0.04μg/L. The 80%, 50%,20% inhibition binding effect dose (IC20, IC50, IC80) of AFB1 were 0.14μg/L, 0.63μg/L and 3.04μg/L respectively. The assay range was 0.1-10μg/L. The cross reactivity with AFB2, AFG1 and AFB2 were 0.7%, 0.5% and 2.3% respectively. The results showed that the AFB1- TRFIA is great method with high sensitivity for detecting AFB1 in sample. The accelerated testing method show that the validity period should be over 1 year when the kit was storaged in 4℃.(3) The factors which could bring errors to sample determination of AFB1 were analyzed and optimized. Effect of saltd density, fat and temperature for ELISA were studied, the result shows that fat has remarkable influence to test. Different sample were extracted by different methods, for powder sample, ultrasonic radiation on extraction for 10 min was best; for liquid sample, shake by machine to extract AFB1 from the sample was more efficient.