| Aflatoxin(aflatoxin,AF)was a secondary metabolite produced by Aspergillus.AF had acute,chronic,carcinogenicity,immunosuppression and other toxic effects on human health.Aspergillus produced AF including B1,B2,G1 and G2 under natural conditions and the 4 kinds of toxins residues had synergistic and additive effects on human health toxicity.The implementation of the total aflatoxins(TAFs)(B1+B2+G1+G2)detection had become the development trend in the field of food quality and safety determination.Compared with the various detection methods of TAFs,GICA technology had the advantages of fast,simple,specific,good stability,can be detected on the spot,large sample size and other advantages and was the main technical means to achieve AFs detection.The purpose of this study was to establish TAFs-Strip method for the detection of TAFs,which provided a reliable technical support for the rapid detection of food TAFs residues.The main contents and conclusions of this study were as follows:1.According to the molecular structure and the active site of AF,4 kinds of B family AF and 3 kinds of G family AF antigen were designed,which were identified by UV,SDS-PAGE and Balb/c mice immunized.The results showed that the OAE method was the best for B family AF artificial antigen synthesis,which the conjugation ratio of AFB1 to BSA was about 8.46: 1 and the AFB pAb of high-titer,sensitivity with 13.6 ?g/kg of IC50 value,specificity with 100% for AFB1 and broad with 100% for AFB2 had been produced in sera of immunized Balb/c mice,which made it possible to establish immunoassay of AFB residues in food.In the same way,the AFG p Ab of high-titer,sensitivity,specificity and broad for AFG had been obtained.2.Balb/c mice were immunized with AFB1-BSA(OAE)and AFG1-BSA(SA),the titre of pAb was detected by indirect ELISA and blocking ELISA.The hybridoma lines that secrete AFB1 mAb and AFG1 mAb were established with using monoclonal antibody hybridoma technology.The AFB1 mAb and AFG1 mAb were induced from in vivo method and and their immunological properties such as titer,affinity,sensitivity and specificity of the mAb were characterized.The results showed that four hybridoma cell linesof 2A11,2F6,3G2 and 3B3 were screened and the 2A11 was the best,the indirect ELISA titer of the mAb were 1?(6.4×102)in supernatant and 1?(5.12×105)in ascites and its Ka was 1.05×109 L/moL.The m Ab of 2A11 showed good sensitivity with an IC50 of 6.22 ?g/kg and hand specificity to AFB1 and had 82.31%cross-reactivity(CR)to AFB2,50.11% to AFG1,12.48% to AFG2.AFB m Ab of high-titer,sensitivity,specificity and broad had been generated and made it possible to establish immunoassay of AFB residues in food.In the same way,AFG mAb of high-titer,sensitivity,specificity and broad had been obtained.Two hybridoma cell lines of 3F10 and 4E7 were screened and the 3F10 was the best,the indirect ELISA titer of the mAb were 1?(1.28×104)in supernatant and 1?(5.12×105)in ascites and its Ka was 1.36×1010 L/moL.The mAb of 3F10 showed good sensitivity with an IC50 of 5.04 ?g/kg and hand specificity to AFG1 and had85.81% CR to AFG2.4.Base on the principle of gold immunochromatography assay(GICA),a strip for detection total aflatoxin(TAFs-Strip)was developed with AFB mAb and AFG mAb and its traits were tested.The results showed that the calibration curve of TAFs-Strip was typical sigmoid curve fitted to the four parameter logistic,its detection limit was 4 ?g/L and its sensitivity was as same as that of competitive HPLC-MS/MS,its coincidence rate was 100% compared with HPLC-MS/MS.The TAFs-Strip possessed rapidity,sensitivity,specificity and briefness and was proved to be used for the rapid test of TAFs residues in animal food. |
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