Study On An Enhanced Quantitative Immunochromatographic Assay For Alfatoxins Detection In Peanuts

Aflatoxins are a group of structurally-related toxic metabolites mainly produced by Aspergillusflavus and Aspergillus parasticus, which distribute in stage of crop growth, harvest, storage andprocessing. Aflatoxins, one of the most toxic natural chemical carcinogens ever found, lead to a seriousthreat to human and animal health, serious economic losses, and even social panic. Aflatoxincontamination has become a focus of concern and difficulty in the field of agro-products and food safety.The rapid quantitative detection technology for aflatoxins in grain and oilseeds products, which is ofgreat significance for the national conditions of the dispersive planting mode in China, can effectivelyprotect the health of consumers in China and improve the international competitiveness for agriculturalexports, especially peanut products in China. In this study, we developed a quantitativeimmunochroamtogrphic assay to determine the occurrence of the aflatoxins in peanuts based on aportable reader developed independently and a quantitative model proposed firstly for goldimmunochromatogrphic assay of aflatoxins detection. The method has the advantages of increasing theaccuracy of quantitative determination and resolving the problem of unacceptable variability betweenstrips due to the dose of gold conjugate. The main conclusions of this study are as follows:1. A portable reader for a rapid quantitative detection of aflatoxins based on goldimmunochromatogrphic assay was developed successfully. The recognition of gray characteristics ofgolden strips consisted of the process of biochemical, optical, electrical and software processingcomprehensively. Using colloidal gold immunochromatographic assay as primary sensor and the CMOSimage chip as a diodesensor, the gray information, which was the qualitative results of the rapidimmunochromatographic assay of aflatoxins detection, was quantified and expressed as imageinformation with the means of the light, machine, electricity, computer and other technologies. Theimage information was processed and analyzed to obtain the quantitative results for the sake of thetechnology of computer vision and modern information processing. Finally, a portable reader for a rapidquantitative detection of aflatoxins based on gold immunochromatogrphic assay was developedsuccessfully for the determination of aflatoxins in peanuts by the quantitative immunochromatographicassay.2. In this study, a quantitative model for gold immunochromatographic assay based on monoclonalantibody was established firstly to increase the accuracy of quantitative determination. Based on thequalitative immunochromatographic assay for aflatoxins developed previously in our lab, with thecolloidal gold-labeled monoclonal antibodies, a quantitative model was established by capturing all theprobe by the excess rabbit anti-mouse IgG on the C line and the antigen on the T line, and a standardfactor of (T+C) was chosen as the appropriate factor to normalize the data and reduce the variabilityamong strips. Making use of the model, a quantitative immunochromatographic assay for aflatoxins inpeanuts was developed successfully. 3. A quantitative immunochromatographic assay for measuring aflatoxin B1in peanuts rapidly wasdeveloped through the portable strip reader developed independently based on the model describedabove. It showed that the differences among strips could be decreased effectively by the establishedstandard curve, which was confirmed by the intra-and the inter-day precision of data (RSD6%-27%) inthree reduplications. The recovery of the study is88%-119%. The LOD in actual peanut samples was0.9μg/kg. The results were confirmed by HPLC with the relative error of less than17%.4. An enhanced quantitative immunochromatographic assay, connected to a portable strip readerdeveloped independently, was developed for the rapid detection of four major aflatoxins (B1, B2, G1andG2) in peanuts based on the above-mentioned model. This one-step assay has enhanced repeatability-aswas confirmed by analysis of the intra-and inter-day precision of the data (RSD7.14%-18.14%) forthree replications. The recoveries for the intra-day and inter-day experiments ranged from71.26%to105.69%and from94.86%to123.2%, respectively. Quantification was obtained using an externalcalibration curve, which can be stored in the strip reader. The limit of detection (0.062μg/kg) and widedynamic range (0.081-20.84μg/kg) allow for the direct assessment of contamination by the four majoraflatoxins in natural peanut samples. Twenty-three peanut samples were analyzed following simplesample preparation. A strong linear relationship was observed (y=0.967x+0.201, R2=0.966) when datawere compared with those obtained by a reference ELISA method.The difficulty that the immunochromatographic assay is hard to meet the demands of rapidquantitative detection has been resolved. And the quantitative lateral flow immunoassay presented heremay be a new approach for effective monitoring of aflatoxins contamination with advantages ofprocedure simplicity, rapid operation, immediate results, low operating and equipment costs and theon-site detection of aflatoxins contamination for local monitoring organizations.