Study On Rapid Immunoassay Test Kits For Aflatoxins In Food And Feed

Aflatoxins are a group of metabolites mainly produced by Aspergillus flavus and Aspergillus parasiticus. Aflatoxins are extremely toxic and carcinogenic, which can cause both acute and chronic toxicity in humans and many other animals. Since they are heat-stable and widely distributed, it's hard to completely avoid or prevent the harmful effects of aflatoxins, even in developed countries with advanced technologies of agricultural production and food processing. Aflatoxin contamination has been one of the great threats to the human health, and has caused great economic losses. Sensitive, accurate, rapid and easy detection technologies and kits have become indispensable tools for the aflatoxin contamination supervision, and have a broad application prospect and important practical significance.Analytical methods for aflatoxins adopted presently are skilled technician requirement, time-consuming and costly, which greatly restrict the wide implementation of aflatoxin contamination monitoring. Because ELISA kits and immunochromatograhpic strips for aflatoxin are easy to use and low-cost, they are favorable in practical applications. However, the convenience and sensitivity of the current products can not fully satisfy the increasing requirements of the market. With the increasing demands, it is important to develop high sensitive, high accurate and easy operated immunoassay technologies and kits for aflatoxins. It is not only for the safety of agricultural products and food, but also to promote the development of agricultural industry and to break the technical barriers to trade. This study aiming at development of monoclonal antibody ?Mab? against aflatoxin B₁?AFB₁? and aflatoxin M₁?AFM₁?, and establishment of rapid detection method for aflatoxin in food, feed and milk. ELISA kits and immunochromatographic strips, which are sensitive, accurate, simple, easy to operate and fast, were also developed for the aflatoxin contamination monitoring.Main research contents and novelties are as follows:1. AFB₁ antigen were prepared, and the molar ratio of the AFB₁:BSA were 14.5:1. Hyridoma cell 2A4 capable of secreting Mab against AFB₁ was obtained by immunizing mice with antigen. The affinity constant of the anti-AFB₁ antibody were 7.9× 10⁹ L/mol. Compared with other published Mabs against AFB₁, the Mab of 2A4 had a better sensitivity and specificity.2. After optimization of coating buffer, pH of assay buffer, concentrations of anti-AFBi monoclonal antibody and HRP labeled AFB₁-BSA, and sample dilution reagent, a one-step indirect competitive ELISA kit for AFB₁ was successfully developed. The limit of detection and the IC₅₀ for AFB₁ was 7.6 pg/mL and 66 pg/mL, respectively, which were much better than others. The linear range was between 10 pg/mL and 810 pg/mL for AFB₁. Spike recoveries for maize, soybean meal and fishmeal samples were ranged from 108.4% to 134.8%, and the intra-and inter-assay relative standard deviations were not greater than 12.8% and 11.6%. The aflatoxin B₁ residues in sixty samples were determined by the ELISA kit and high performance liquid chromatography-tandem mass spectrometry ?HPLC-MS/MS? respectively, and the result of ELISA kit was consistent with that of HPLC-MS/MS. The ELISA kit could be used for the detection of AFB₁ residues in foodstuff and feed.3. After optimization of some parameters of ELISA method, an ELISA kit for AFB₁ in infant supplementary food was successfully developed. The limit of detection and the IC50 for AFB₁ was 5.5 pg/mL and 24 pg/mL, respectively. The linear range was between 5 pg/mL and 100 pg/mL for AFB₁. Spike recoveries for nutrition rice powder, soybean powder and soybean flour samples were ranged from 84.1 to 95.2%, and the intra- and inter-assay relative standard deviations were less than 9.3% and 7.4%. The limit of detection for AFB₁ residues in samples met the requirements of aflatoxin contamination supervision in China and the European Union.4. For the detection of AFB₁ in oil by traditional methods, complex pre-treatment process were needed such as extraction and purification, which was hard to use and cost lots of organic solvents. A "green", simple, fast and sensitive method for detection of AFB₁ in plant oil was creatively developed based on colloidal gold immunochromatographic assay. The determination of AFB₁ residue in oil could be achieved simply by mixing oil and water in indicated ratio and then loaded onto the strip for visual detection. The visual detection limit of test strip for AFB₁ residue in oil sample could reach to 1.5 ?g/kg, and could be tuned to match different AFB₁ regulations simply by changing the water/oil ratio. The accuracy of this method was validated by Chinese national standard method. Because it is very easy to operate and no organic solvent was needed, it could be utilized for oil safety control in home by people without professional training. This method can be used for the rapid determination of AFB₁ residue in oil in laboratory and at home, and has a broad application prospect.5. Hyridoma cell 2F12 capable of secreting Mab against AFM₁ was obtained by injected mice with AFM₁ antigen. The affinity constants of 2F12 Mab were 1.8 ×10⁹L/mol and had a high cross reactivity with AFB₁. After optimization of some parameters of ELISA method, an ELISA kit for simultaneous detection of AFM₁ and AFB₁ in milk and milk products was successfully developed. The limit of detection and the IC₅₀ for AFM₁ is 37 pg/mL and 211 pg/mL, respectively. The linear range was between 50 pg/mL and 1500 pg/mL for AFM₁, Cross reactivities for AFM₁ and AFB₁ were 100% and 102%, and less than 20% for the other analoges. Spike recoveries for milk and milk products were ranged from 87.9 to 109.1%, and the intra- and inter-assay relative standard deviations were not greater than 10.8% and 11.1%. The results of this kit were consistent with that of imported commercial ELISA kits.