Study On The Immunoassay Technology For Aflatoxin G

The specific antiserum was produced by immuning the Balb/c mice with AFG1-BSA synthesized in the lab. After four times,immunization, the interested spleen cells were selected to fuse with the SP2/0 cells and one positive hybridoma cell was screened against AFG1. The monoclonal antibody that secreted by hybridoma cell was purified and characterized through the indirect competitive ELISA, which showed high specific with AFG1and made a foundation for the immunoassay of afltoxin G. The main research contents and results were as follows:(1) With the AFG1 as the raw material, the artificial antigen of AFG1-BSA was synthesized by reducing alkylation. The conjugate structure was identified by UV scanning and fluorescence analysis. The results showed that it had the different characteristic absorption peaks and stronger fluorescence value compared to AFG1. The titer of antiserum could reach 1:128000 after four times immunization and IC50 was 14.7ng/mL through the indirect competitive ELISA.(2) The positive spleen cells were selected to fuse with SP2/0 cells, one hybridoma cell line was obtained through the screening culture of HAT hemi-solid medium which was named 2G6, and cultivated in liquid culture medium for detecting the stability for three months. The characteristise of monoclonal antibody were identified. The results of the indirect competitive ELISA showed that the subtype was IgG2a, IC50 was 17.18ng/mL, the cross reaction with AFG2 was 87%; and the cross reaction with AFB1,AFB2,AFM1 was less than 5%. The antibody showed the high specify with aflatoxin G, especially for G1 .(3) With 2G6 monoclonal antibody obtained, the method of indirect competitive ELISA for AFG1 was established. On the basis of our laboratory, six factors were optimized including the concentration of coating antigen, antibody, methanol, blocking, dilution and the salt ionic and determined the optimum conditions as follows: coating antigen 2μg/mL, antibody 1:32000, blocking 1%OVA solution, 20% methanol/water,PBST and 0.14M salt ionic. After the optimization, the sensitivity reached the highest, IC50 for AFG1 was 17.18ng/mL±3.5 ng/mL, the lowest detection limit was 0.6018ng/mL±0.021ng/mL.(4) A spiking technique was applied in peanuts with three concentrations: 5ng/mL,50ng/mL and 200ng/mL, and the samples were extracted with 80% methanol (v/v) containing 4%(w/v)NaCl. The recoveries were 111.4%,94.18% and 102.8%, respectively. These results suggested that the established ELISA method could be used for the rapid and sensitive determination of aflatoxin G in agri-food.