| Aflatoxins are a group of extremely toxic metabolites produced by some Aspergillus species namely A. flavus and A. parasiticus. Toxigenic funguses distribute on each level of food chain, and the harm of aflatoxins can not be absolutely avoided or prevented even in developed countries with advanced technologies of agricultural production and food processing. Aflatoxin is one of the strongest carcinogens known up to date. In the past years, malignant events of poisoning death caused by aflatoxin constantly emerged, which not only seriously threatened the health and life safety of human and animals, caused a great loss of economics, but also easily resulted in panic of public and seriously affected the construction of harmonious society. Therefore, aflatoxin contamination has become one of the difficult problems of general interest in agro-product quality and food safety field and been paying great attention by countries all over the world. Besides, recently European Union, etc, set up rigorous technical trade barriers to exportation agro-products of China by establishing rather strict regulation of allowable limit standards for aflatoxins. Exported agro-products especially cereal and oilseeds products of China are continuously rejected, detained, returned or even destructed due to the exceeding aflatoxin content, which bring enormous economic loss to China.Therefore, sensitive, accurate and rapid test technologies become indispensable for the aflatoxin contamination supervision and ensuring of agro-product secure consumption. Aflatoxin analytical methods adopted presently are skilled technician requirement, time-consuming and costly, which greatly restrict the wide implementation of aflatoxin contamination monitoring. Besides, the lack of key reagent of high-quality monoclonal antibody (MAb) also becomes a vital problem for the establishment of rapid test technologies. To protect the health and life safety of human and animals, decrease adverse influences of trade barriers, a series of MAbs against aflatoxin B1,M1 and total aflatoxins, respectively, were prepared for the urgent problems. Based on these MAbs, associated rapid test technologies for aflatoxin contamination in foodstuff, oilseeds, milk products and so on were established and, correspondingly, matching aflatoxin strips were also prepared. What's more, an aflatoxin digital strip was firstly developed. The study provided sensitive, accurate, simple, rapid and low cost methods for monitoring of aflatoxin contamination in cropping staple agro-products (e.g., cereal and oilseeds) and milk products. Main research contents and novelties are as follows:1. An ultra-sensitive anti-aflatoxin generic MAb and MAbs with highly specific to aflatoxin B1 or M1 were prepared. High-quality antibody is one of the key factors for successful establishment of any immunological method. In order to prepare high-quality anti-aflatoxin antibodies, regular semisolid medium, stem cell medium and liquid medium culturing methods were successively adopted to select and breed excellent monoclonal hybridoma cell lines. A novel two-step screening method was established based on the previous research. Finally, an ultra-sensitive anti-aflatoxin generic MAb (1C11) was screened out using the novel two-step screening method. The sensitivities (expressed by IC50) of MAb 1C11 to aflatoxinB1, B2, G1 and G2 were 1.2,1.3,2.2 and 18.0 pg/mL, respectively. Compared with those corresponding anbodies reported home and abroad,1C11 was the most sensitive generic MAb for aflatoxins up to now. Meanwhile, another two high-quality hybridoma cell lines 3G1 and 2C9 were selected out using stem cell medium culturing method. MAbs secreted from 3G1 and 2C9 showed high specificity to aflatoxin B1 and aflatoxin M,, respectively. MAb 3G1 showed 100%,6.4%,<0.02% and <0.02% cross-reactivities toward aflatoxin B1, B2, G1 and G2, respectively, MAb 2C9 showed no cross-reactivity to the above four aflatoxins. Comparing comprehensively specificity and sensitivity with corresponding anbodies reported home and abroad, it was found that 3G1 and 2C9 were both the best MAbs against single aflatoxin so far.2. A high specific immunochromatographic strip for aflatoxin B1 was developed and showed the highest specific compared with the present aflatoxin B1 strips. Visual detection limit (VDL) of the immunochromatographic strip test (IST) to aflatoxin B1 was 1 ng/mL, and the IST showed no cross-reactivity to aflatoxins B2, G1 and G2, The IST was applied and validated by high performance liquid chromatography (HPLC) in the detection of aflatoxin B1 contamination level in 62 samples which belonged to 7 types of agro-products, the results showed that the coincidence rate of two methods was up to 98.39%(61/62) indicating that the developed strip could well meet the requirement of sepcific detection for aflatoxin B1 in agro-products and food samples.3. A high specific and sensitive immunochromatographic strip for aflatoxin M1 was developed. VDL of the IST to AFM1 in milk detection solution was 0.3 ng/mL. The strip was applied for milk and milk power, and the results were compared with an indirect competitive ELISA. The results showed that the coincidence rate of IST and the ELISA was up to 100%, indicating great application potential of the developed strip for aflatoxin M1 contamination monitoring in milk and milk products.4. An immunochromatographic strip for total aflatoxins was firstly developed. VDLs of the IST for aflatoxin B1, B2, G1, and G2 were 0.03,0.06,0.12 and 0.25 ng/mL, respectively. Compared with single aflatoxin ISTs reported home and abroad, the sensitivity of developed IST to each aflatoxin was the highest up to now. With HPLC for reference, the IST was applied in the detection of aflatoxin contamination in 64 samples which belonged to 7 types of agro-products, the coincidence rate of the results between IST and HPLC was 95.31%(61/64). The detectable concentrations of IST for aflatoxin B1, B2, G1, and G2 in real samples were 0.23,0.45,0.90 and 1.88μg/kg, respectively, which were far lower than the limit standard levels set by European Union. Therefore, the developed strip could be well applied in the contamination monitoring of total aflatoxins in real samples.5. Digital immunochromatographic strip for aflatoxins was firstly developed. A model of the digital immunochromatographic assay was firstly constructed, taking aflatoxin as the target analyte, the model was applied, and an aflatoxin digital strip was prepared. Taking aflatoxin B1 detection in real samples as an exemple, the digital strip could provide five contamination levels' detection of <0.6 ng/mL,0.6～1.25 ng/mL,1.25～5.0 ng/mL,5.0～20 ng/mL and >20 ng/mL, and the concentration range of aflatoxins could be red out by naked eyes within 15 min. With HPLC for validation, the digital strip was applied in the detection of aflatoxin B, contamination level in peanut and feedstuff samples, the coincidence rate of detection results between two methods was up to 100%(31/31). the results showed that the designed and constructed digital immunochromatographic assay could solve the technical difficulties that current immunochromatographic assay can not meet visual quantitative rapid detection. The study provided a novel way for quantitative immunochromatographic assay and a powerful technical support for developing digital strip of other target analytes.
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