Preparation And Immunoassay Application Of Antibodies For Mycotoxins In Food

Mycotoxins are toxic secondary metabolites which are produced by filamentous fungis.Except of the acute toxicity,mycotoxins also have the carcinogenic,teratogenic,mutagenic effects and that pose a great threat to human survival and health.The toxigenic fungi can infect cereals and fruits during the whole chain of food production and produce mycotoxins as grow,harvest,storage,processing and even transportation stage.Because these mycotoxins were difficult to remove during the food processing,the mycotoxin contamination is considered to be unavoidable and unpredictable.As the great harm of mycotoxin contamination,strict limits standard on mycotoxins were developed by different countries in the world and various detection methods were developed for mycotoxin determination.As the cheap,convenient,fast and easy operation method,the immunologic technology becoming an important method for the mass sample screen and on-site rapid detection.In this research,several main mycotoxins were selected as objects as the aflatoxins,deoxynivalenol,ochratoxin A,fumonisins,sterigmatocystin,citrinin and alternaria toxins.The corresponding high sensitivity and high affinity monoclonal antibodies were prepared and be used for the development of rapid immunology detection methods.(1)Haptens were obtained by the derivation of mycotoxins firstly or mycotoxins directly conjugated with carrier proteins to get the complete antigens and confirmed by UV-spectroscopy,mass and electrophoresis.(2)Through the mice immunization with complete antigens,spleen cell fusion,hybridoma screening and sub-cloning,ascites preparation and purification,11 special/group selection cell lines were obtained for target mycotoxins.(3)All the special monoclonal antibodies were characterized and 9 ELISA methods were developed after the optimization and verified by the recovery experiment.The antibody subtype,affinity constant,IC50 value and recovery rate for each m Ab were: AFB1(7A1),Ig G1,6.72×109 L/mol,0.013 ng/m L,and 76.0~ 93.4%;AFM1(2E7),Ig G2 a,5.31×109 L/mol,0.023 ng/m L,and 73.3 ~91.5%;DON(1G3),Ig G1,4.26×109 L/mol,1.05 ng/m L,and 89.6~114.5%;OTA(1G1),Ig G2 b,4.95×109 L/mol,0.07 ng/m L,and 72.5~ 114.0%;FB1(1H1),Ig G2 a,7.71×109 L/mol,2.17 ng/m L,and 89.5~ 111.5%;STG(4G10),Ig G2 a,3.72×109 L/mol,0.09 ng/m L,and 78.3~102.5%;CIT(1F2),Ig G2 b,1.65×109 L/mol,0.76 ng/m L,and 117.8~121.8%;AOH(1G4),Ig G1,4.71×109 L/mol,3.49 ng/m L,and 97.5~114.4%;Te A(5F6),Ig G2 b,3.29×109 L/mol,280.3 ng/m L,and 89.3~ 115.8%.(4)The special m Abs were selected for the preparation of colloidal gold test strip.After the optimization,for AFB1,AFM1,DON,OTA,FB1,STG,CIT,AOH and Te A,the visual limit of detection,cut-off value and calculated limit of detection for each mycotoxin were0.25,0.1,10,5,2.5,3,4,160,800 ?g/kg;0.5,0.2,50,20,10,12,20,640,3200 ?g/kg;0.04,0.02,3.79,1.26,0.53,0.58,0.88,19.4,206.6 ?g/kg.(5)The group selection m Abs were selected for the preparation of the multiple-detection colloidal gold test strip for twenty kinds of mycotoxins.The visual limit of detection,cut-off value and calculated limit of detection for ZENs were 0.1~1,0.25~2.5,and 0.04~0.17 ?g/kg;for DONs were 2.5~250,5~500,and 0.06~49 ?g/kg;for T-2 and HT-2 were 0.5~1,1~10,and 0.15~0.22 ?g/kg;for AFs were 0.25~1,0.5~2.5,and 0.056~0.49 ?g/kg;for FUMs were 2.5~10,5~25,and 0.53~1.05 ?g/kg.