Study Of Aflatoxin M1Immunological Rapid Detection Method

Recently, fungaltoxin contamination has become one of the main contaminations in the food contamination, especially Aflatoxin contamination. Aflatoxin includes a variety of different species that all have particularly strong toxiceffect, particularly Aflatoxin M1which extensively distributes in milk ormilk products. Aflatoxin M1is closely related to people’s daily life, and is frequently concerned.Aflatoxin M1is the in-vivo metabolic product of Aflatoxin B1and is a colorless, rectangular and flake crystal. Its melting point is299℃, and it can emit can emit violetfluorescence under UV light365nm. Aflatoxin M1has strong toxicity; if people ate foods which contain AflatoxinM1, it is likely lead to functional failures in the liver, liver cancer or death directly. There are many current detection methods for Aflatoxin M1, such as thinlayer chromatography (TLC), high performance liquid chromatography (HPLC), LC-MS/MS method and so on. The above methods have several disadvantages and limits, such as the cumbersome sample pre-treatment, expensive equipment, demanding operation skills, low sensitivity, and incorrect results and so on. The immunological detection method, based on antigen-antibody reactions, has a variety of advantages, for instance high sensitivity and specificity, simple operation skills andsatisfies the field massive detection and so on. Therefore, it is urgent to establish the fast detection method for Aflatoxin M1which is based on immunology.In this study, we should try our best to development of rapid detectionkits for AFM1detection.The main contents and results of this study were as follows:1. Synthesis and identification of AFM1artificial antigensVia Oximation method to link carrier proteins BSA and OVA to AFM1, immunogen AFM1-BSA and coating antigen AFM1-OVA were synthesized; then they were identified by TLC,UV scanning and SDS-PAGE.The mice polyclonal antiserum was obtained by immunizing mice and then identified by ELISA method.The results showed that the migration distances of oximation products in TLC plates were shorter; absorbance peak of AFM1-BSA appeared in274nm, and did not overlap with that of BSA or AFM1; the migration speed of AFM1-BSA was significantly smaller than that of BSA; the antiserum titres of all the three Balb/c mice were all about1×10-4, particularly the antiserum of Balb/c mice No.3that has the best sensitivity and IC50of133.8ng/mL.2. Preparation of monoclonal antibody against AFM1and establishment of immunological quantitative detection methodIn this study, based on the mice polyclonal antibody of AFM1, cell fusion technology and hybridoma technology, AFM1-BSA was obtainedsuccessfully and proved to be high affinity, sensitivity and specificity. Two hybridoma cell lines of2C4E3and2C6F2were screened and detected via ELISA method, the titers were1:1.28×103and1:3.0×102in supernatant respectively;2C4E3was selected for expanded cultivation, the titer of ascitesobtained from mice was1:1.512×105of2C4E3in ascites; after calculation, the affinity constant (Ka) was2.71×1010L/moL, Standard inhibition curve was y=－0.4039x＋1.9463with R2=0.9872，IC50=3.8ng/mL; The rates of cross reaction with other compounds were lower than1%.3. Development of rapid detectionkits for AFM1detectionBased on the enzyme-linked Immunosorbent assay, we obtained that the optimum concentration of coating antigen was1:1000and the maximum working concentration of antibody was1:32000, finally the ELISA detection method and the rapid detection kits for AFM1detection (AFM1-Kit) were developed with AFM1mAb.The indicators of AFM1-Kit all met the expectations; the correlation coefficient of regression equation (R2) was0.9872; the recoveries were above80%; the coefficient variation were below15%, and the rates of cross reaction with other compounds were lower than1%. The AFM1-kit has many advantages such as stable performance, fast, specificity; it can be widely applied in food production.