| Aflatoxin is found in most nuts,cereals and cooking oils.Residue of veterinary drugs,such as clindamycin(CLIN)and lincomycin(LIN)are often detected in animal derived foods.CLIN and LIN not only elicit nausea and injecting pain,but also increase the risk of pseudomembranous bowel cancer.Among the family of Aflatoxin,carcinogenic Aflatoxin B1(AFB1)is the most toxic and frequently detected.Listed below is our research plan.1? Preparation of CLIN monoclonal antibody(m Ab)CLIN and LIN immunogen and inclusion precursor were prepared by succinic anhydride method,activated ester method and sodium periodate reduction method.The coupling ratio was 3.9 to 6.7 by mass spectrometry.After mice had been immunized with CLIN-BSA conjugate,the specific antibodies were detected in all mice.The mouse with the highest antiserum sensitivity and the highest titer was used to produce the cell line 3B9 that secreted m Ab steadily.Based on the monoclonal antibody prepared by 3B9,an indirect competitive ELISA method was established.The antibody working concentration of this method is 0.096 ?g/m L,the working concentration of the coated antigen is 0.12 ?g/m L,the measured ODmax is 1.795,and the IC50 of CLIN is 1.2 ng/m L.A distinct specificity of m Ab towards LIN was found with a cross reaction rate of 25%,and IC50 value of 5.8 ng/m L.2? To establish an indirect competitive ELISA protocol for detection of residual CLIN and LIN in beef,chicken,pork and fish: Under optimized conditions,the standard curves of the ELISA method for CLIN and LIN were obtained.The IC50 values of the standard curves are 0.3 ng/m L(CLIN)and 1.2 ng/m L(LIN),respectively.The matrix effect was basically eliminated in 10 minutes by an optimized pretreatment,which enable detection of residues of CLIN and LIN in beef,chicken,pork and fish.The detection limits of this ELISA method were 1.8 ?g/kg(CLIN)and 6.8 ?g/kg(LIN)respectively.In the recovery test,the average recovery rate ranged from 76% to 112%,and the intra/inter assay coefficient of variation was between 7.1% and 13.2%,no more than 15%.3? Establishment of an indirect competitive ELISA method for detection of AFB1 residues in rice and rice flour: An indirect competitive ELISA method was established for the food substrates such as rice and rice flour,which were widely used in the south of China.New method is credited for high specificity and recognition level of AFB1 with IC50 value of 0.400 ng/m L.This was demonstrated by negligible recognition of other kinds of mycotoxins,whose cross reaction rate was below 1%.In this study,a sample pretreatment method for the rapid detection of AFB1 residues in rice and rice flour was established.Sensitivity of detection by the present ELISA method has reached 0.89 ?g/kg AFB1 using rice and rice noodles as models.The average recovery range is 73.9% to 102.7% in the added recovery experiment,and the CV of batch to batch is within acceptable range from 5.16% to 11.36%. |
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