High-sensitivity, High-throughput And Rapid Detection Immunoassay For Mycotoxins

Mycotoxins are pathogenic and fatal secondary metabolites which are produced by somefungi during growing. Mycotoxins could be fabricated during the production, storage,processing and circulation of cereal in case of improper conditions. Because of the acutetoxicity as well as the ability of carcinogenicity, teratogenicity and mutagenicity, all kinds ofdetection methods were developed. With advantages in high sensitivity, rapid, highthroughput and more residues and field test, immunoassay has been one of the importantmeans of rapid screening and detection mycotoxin detection. In this research completeantigens were prepared by conjugating the haptens of mycotoxins to the proteins carrier, andthen were used to immunize mice. The cell lines of monoclonal antibodies (mAbs) thatagainst AFB1, AFM1, OTA, ZEN, DON and T-2toxin were obtained through cell-screening.Rapid assay methods for the six mycotoxins were developed based on the mAbs. The maincontents are as follows.(1) The haptens were obtained by deriving the mycotoxins, and confirmed byUV-spectroscopy and mass. Subsequently complete antigens were obtained by conjugatingthe haptens with protein carriers. Finally eight cell lines were obtained through immunization,cell fusion and subclone.(2) Characteristics of mAbs were identified, the affinity constant was AFB14.2×109L/mol(11A9) and2.29×109L/mol (5C3);AFM11.28×109L/mol (G3D9) and2.29×109L/mol(H1);OTA1.09×109L/mol;ZEN2.26×109L/mol; DON1.6×108L/mol; T-2toxin1.35×108L/mol, respectively. Antibody subtypes and cross reaction were also measured.(3) Some conditions were optimized, such as the concentrations of coating antigens andantibodies, the pH, ion strength and methanol concentration of the standard solutions, reactiontime of primary antibodies, Secondary antibodies and colour developing. Under the bestconditions, the IC50were0.043±0.003ng/mL(AFB1,11A9)and0.025±0.005ng/mL(AFB1,5C3),0.032±0.006ng/mL(AFM1, G3D9) and0.035±0.005ng/mL(AFM1, H1),0.092±0.006ng/mL (OTA),0.054±0.004(ZEN) ng/mL,31.60±2.89ng/mL(DON),0.431±0.019ng/mL(T-2toxin), respectively.(4) The ELISA methods for six mycotoxins were developed on the base of obtained eightmAbs. The ELISA methods were confirmed by spiked assay. The recoveries were between80%~120%, and the differences between group and within group were less than20%.(5) F(ab')2fragment of AFB1mAbs were prepared by optimizing the enzymatichydrolysis conditions of antibodies. Anti-idiotype antibody was obtained by immunizingrabbits, and was used to develop nontoxic ELISA kit. The kit was confirmed by spiked assay.The recoveries were between110%~130%, and the differences between group and withingroup were less than10%.(6) The rapid field-test strips for detecting AFB1, AFM1, OTA, ZEN, DON andT-2toxinwere developed. The LOD is0.2,0.1,0.2,1,50,5ng/mL and cut-off level were0.5,0.2,1.0,2.0,100,10ng/mL, respectively. (7) The muitiple rapid field-test strips for simultaneously detecting ZEN?T-2?AFB1andOTA was developed. The LOD is2,16,1,2ng/mL and cut-off level were4,32,2,4ng/mL,respectively.