Development Of Novel Immunoassays For Aflatoxin In Agro-products

Aflatoxins are a group of extremely toxic and carcinogenic small molecular metabolites produced by certain Aspergillus species, namely A. flavus and A. parasitic. Aflatoxins have been implicated as causative agents in human hepatic and extrahepatic carcinogenesis. Because of their great harm, the International Agency for Research on Cancer(IARC) has classified aflatoxins B1, B2, G1, and G2 into group I human carcinogens. Aflatoxins frequently contaminate a wide range of agri-foods and animal feeds. Outbreaks of acute aflatoxin poisoning have become a recurrent public health issue. Therefore, to promptly develop new and reliable analytical technique for these carcinogenic compounds has become an important requirement of food consumption safety to meet food safety concerns.In recent decades, many methods have been developed for aflatoxin determination, such as high performance liquid chromatography(HPLC), liquid chromatography–tandem mass spectrometry(LC-MS/MS), and immunoassay. Among these methods, immunoassay has been regarded as a valuable supplement to chromatographic techniques due to its advantages in sensitivity, specificity, and cost saving. In recent years, the immunoaffinity assay, nanogold labeled immunochromatographic assay, and time-resolved fluorescent immunochromatographic assay had been developed in our lab for aflatoxin detection in agro-products.With the innovation and progress of technology, some new immunoassay methods have been developed constantly. Such as label-free immunoassay, it requires only one reaction step all over the assay procedure and achieves a direct and short-time consuming determination; immuno-polymerase chain reaction(IPCR) is also a novel method whcih combines the versatility of ELISA with the sensitivity and signal amplification capability of PCR, it not only leads to higher sensitivity as compared to conventional ELISA, but also reveals a much wider linear range; loop-mediated isothermal amplification(LAMP) is also a novel nucleic acid amplification technique which developed in recent years, it’s has higher specificity and amplification efficiency as compare to conventional PCR, and it’s simple and no special equipment required.Based on an anti-aflatoxin monoclonal antibody 1C11 and an anti-idiotypic nanobody-phage V2-5, we developed three novel immunoassays, which provide new ideas and technology platforms for aflatoxin detection in agri-products. Main contents and innovations of the research are as follows:1. A simple, direct and label-free immunoassay was proposed based on a specific immune binding reaction induced fluorescence quenching of the analyte. Aflatoxin B1(AFB1) was taken as the model analyte, whose intrinsic fluorescence was quenched by the specific anti-AFB1 monoclonal antibody. This immunoreaction-induced fluorescence quenching was utilized to quantitatively determine AFB1. The fluorescence of AFB1 was enhanced by adding 2, 6-Di-O-methyl-β-cyclodextrin, and then the specific anti-AFB1 antibody 1C11 was added into the mixture to perform immunoreaction. The fluorescence scanning results validated that the fluorescence intensity of AFB1 decreased sharply after the immunoreaction with 1C11 in the solvent of 10%(v/v) methanol-water. Based on the optimized parameters, this direct and label-free immune technology was used to quantitatively analyze AFB1. A standard quenching curve for AFB1 standard solution was established, the calibration curve showed good linearity(R2 = 0.9998) for AFB1 from 1 ng/m L to 20 ng/m L. The LOD for AFB1 was determined as 0.35 ng/m L. The results obtained by AIFQ and HPLC assay indicated that the recoveries of AIFQ method were consistent with those from HPLC, showing good application of this AIFQ method in peanut sample analysis. In a direct and label-free way, this fluorescence quenching phenomenon on the target analyte with intrinsic fluorescence signals could be widely employed in determination of AFB1 and other analytes. On one hand, this method is significantly simple and time-saving compared with conventional immunoassay methods. On the other hand, this method is environmentally-benign as it does not need highly toxic antigens, which is also friendly to operators and reduces detection costs.2. A real-time immuno polymerase chain reaction(IPCR) assay was developed for the accurately quantitative detection of aflatoxins in agri-products base on a M13 phage containing aflatoxin anti-idiotypic nanobody and its encoding DNA which was used to design the specific primers. This newly developed assay can integrate the versatility of ELISA with the sensitivity and signal amplification capability of PCR. The limit of detection(LOD) of the assay is 0.02 ng/m L, which exhibits a 4-fold improvement over traditional phage ELISA. Real-time PD-IPCR analysis also revealed higher sensitivity and a wider linear range when compared with our previous research. The developed method was successfully validated with the samples of corn, rice, peanut, and feedstuff, which are major aflatoxin-contaminated agri-products. And the recoveries were from 77.05-122.16%. For further validation, the developed assay was also compared with a reference HPLC method for the analysis of aflatoxins in corn and peanuts, and concordant results(R2=0.991) were obtained. This study demonstrated that phage-displayed anti-idiotypic nanobody could be an excellent reagent to develop a novel real-time PD-IPCR assay with high sensitivity for detection of small molecules. This is the first time that the anti-idiotypic nanobody based real-time PD-IPCR assay has been applied in detection of aflatoxins in agricultural samples. Besides, taking advantages of the unique characteristics of the recombinant phage, there is great potential to develop a multiplexed detection platform for simultaneously detecting multiple analytes in agri-products.3. An immuno-loop-mediated isothermal amplification assays(i LAMP) were developed by using antibody 1C11 and nanobody-phage V2-5. LAMP is performed in a 0.2 m L PCR tube that were placed in a water bath using a regular laboratory water bath, which is kept at 63 oC for 40 min and then heated at 95 oC for 2 min to end the process. The reaction is carried out in a volume containing d NTPs, betaine, Mg SO4, p H8.8 Tris-HCl, KCl,(NH4)2SO4, Tween-20, DNA template, a set of four specially designed primers(B3, F3, FIP, BIP) and Bst DNA polymerase. The assay was used to detect aflatoxin B1, B2, G1, G2, with LOD of 0.1 ng/m L, 0.1 ng/m L, 0.2 ng/m L, 1 ng/m L. The i LAMP consisted of the competitive immunoreactions coupled to the LAMP reaction for detection. This method provides positive results in the visual color of violet, while a negative response results in a sky blue color; therefore, the i LAMP allows to judge the detection result by naked eye. We validated the i LAMP by detecting aflatoxins in peanut and the detection results were consistent with HPLC. In conclusion, the i LAMP is a simple, rapid, sensitive, and economical method for detecting aflatoxin in agri-products with no instrumental requirement.