Preparation Of Monoclone Antibody And Establishment Of Immunological Rapid Test Method Of AFB₁

The study were prepared for AFB₁ of artificial immunogen and coating of the original, obtain its mice polyclonal antiserum by immunizing with AFB₁-BSA;then we abtained AFB₁-hybridoma cell,through cell fusion by the immunized Balb/c mouse spleen cells with SP2/0 fusion.We selected three cell lines monoclonal anti AFB₁ by competitive ELISA and indirect ELISA, selected one line for an expanded system of ascites by mice,and then it was identifide for immunological characterization with the better results of its specificity, the higher sensitivity, lower of IC50, eventually development of rapid determination of AFB₁ residue method.The main contents and results of this study were as follows:1. Synthesis and identification of the artificial antigen for AFB₁Immunogen AFB₁-BSA and coating antigen AFB₁-OVA were synthesized using NHS and EDC by linking carrier proteins BSA and OVA to AFB₁ and identified by ultraviolet scanning,SDS-PAGE.The mice polyclonal antiserum was obtained by Balb/c mice immunization and was identified by indirect ELISA and blocking ELISA.The results showed that the hapten AFB₁ was successfully linked to carrier proteins.Indirect ELISA showed a high titre above 1×10-4 of the antiserum of all the six Balb/c mice. The sensitivity of antiserum of No.3 mice was the best,which IC50 was 48.6 ng/mL.Artificial antigen AFB₁-BSA and AFB₁-OVA were synthesized and sensitivity AFB₁ polyclonal antiserum has been generated in this study, which laid a foundation base for the preparation of monoclonal antibodies and rapid test reagent against AFB₁.The rate of cross reaction of AFB₁mAb with AFB2 was 4.72%, and there was no cross-reactivity to other compounds. AFB₁mAb of high-titer, sensitivity and specificity had been generated.2. Development of monoclonal antibody against Aflatoxin B₁ and establishment of immunology quantitative ELISAOne Balb/C mice were immunized with AFB₁-BSA,The titre and sensitivity of polyclonal antibody was detected by indirect ELISA and blocking ELISA, so as to select the mouse used in cell fusion. AFB₁mAb was prepared by hybridoma technology. The titer, affinity, sensitivity, specificity and subtype of the mAb were characterized. Massive AFB₁mAb were induced from in vivo method.The results showed that the hapten AFB₁ was successfully linked to carrier proteins by the UV scanning spectrum and SDS-PAGE electrophoresis. There hybridoma cell lines of 2H5-F6,2H5-C9,2H9-C3 were screened for specificity to AFB₁, all the isotypes of the mAb were IgG1. The indirect ELISA titer of the mAb were 1:2.0×102 ¹:1.28×103 in supernatant, 1:1.28×105 of 2H5-F6 in ascites, and the affinity constant(Ka) was 2.65×1010L/moL, the mAb of 2H5-F6 showed good sensitivity with IC50 of 0.58ng/mL to AFB₁. The rate of cross reaction of AFB₁mAb with AFB2 was 4.72%, and there was no cross-reactivity to other compounds.AFB₁mAb of high-titer, sensitivity and specificity had been generated, it is possible to establish immunoassay of AFB₁ residues in food.3. Development of rapid determination of AFB₁ residue methodBased on the enzyme-linked immunosorbent assay and Colloidal gold principle, a competitive ELISA kit for determination of AFB₁(AFB₁-Kit) was developed with AFB₁mAb.The calibration curve of AFB₁-Kit was typical sigmoid curve fitted with logistic equation, the detection limit of AFB₁ IC50 was 0.58ng/mL. The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15%.The recoveries from peanut and corn were 92.5% and 95.3%, respectively, when 0.1, 1, 10, 100ng/mL AFB₁ were spiked. The validity of AFB₁-Kit of high-titer, sensitivity and specificity, test strip is very useful for the quantitative, semi-quantitative or qualitative detection of AFB₁ residues.