The Study Of Immunochromatographic Assays For Detection Of Aflatoxin M1

AFM1 was first discovered by Alleroft in 1963 and was named AFM1 in 1965.AFM1 was a highly toxic substance.In 1993,AFM1 has been classified as a Group ? carcinogenic agent by the International Agency for Research on Cancer of WHO.The toxicity and carcinogenicity of AFM1 behind AFB1,and AFM1 is mainly found in milk and dairy products.AFM1 is stable and thermotolerant,it could not be removed completely in the actual milk processing.Governments have formulated strict limits to ensure the safety of dairy products.A one-step immunochromatographic assay(ICA)with two cutoff values was developed to satisfy the European limit standard and China limit standard in this study,it provides a new idea for food administration in the field of AFM1 onsite detection.Meanwhile,the performance of fluorescent microsphere ICA(FM-ICA)and quantum-dot submicrobead ICA(QB-ICA)was systematically and comprehensively compared in quantitative detection of AFM1 in milk,it serves as a reference for selecting appropriate fluorescent labels for the ICA of AFM1.Contamination of AFM1 in milk has two standard limits,namely,the European standard set up at 0.05 ng/m L and the other one(including China,America,and other countries)set up at 0.5 ng/m L.In the second chapter,a one-step ICA with two cutoff values was developed to satisfy these two standardlimits,wherein test zones including test 1 and 2 lines(T1 and T2)were formed by dispensing detection antigen onto a nitrocellulose membrane.The migrating AFM1 analyte in the sample and detection antigen immobilized on two test lines competed for the limited binding sites of AFM1 antibody,which conjugated with colloidal gold.Three red lines appeared at T1 and T2 lines and control(C)line when the sample was negative or the AFM1 analyte concentration was lower 0.05 ng/m L.However,only two red lines appeared at T1 and C lines when the AFM1 analyte concentration was 0.05 ng/m L or higher but lower 0.5 ng/m L.One red line appeared at C line when AFM1 analyte concentration was 0.5 ng/m L or higher.Results showed that the novel ICA was superior to traditional ICAs because of satisfying two standard limits at one step and lower LOD(0.05 ng/m L).Recent years,FM and QB gradually replace the colloidal gold and became a new label of ICA with the advantages of high sensitivity,good accuracy,and high stability.In the third chapter,the performance of FM-ICA and QB-ICA was systematically and comprehensively compared in quantitative detection of AFM1 in milk.The advantages of FM-ICA include lower limit of detection of 42.3 pg/m L with better accuracy,precision,reliability,and practicability under optimum conditions: label p H 7.0,detection time 15 min,1.0 mg/m L of detection antigen on the T line and 0.1 ?L of FM-m Ab.The advantages of QB-ICA include shorter detection time,lower monoclonal antibody consumption(0.01 ?g per ICA test strip)and lower detection antigen concentration(0.1 mg/m L)under optimum conditions: label p H 6.0,detection time 35 min,0.25 mg/m L of detection antigen on the T line and 2.2 ?L of FM-m Ab.The two ICAs were consistent with liquid chromatographytandem mass spectrometry.This study serves as a reference for selecting appropriate fluorescent labels for the ICA of AFM1.