Preparing Of Hybr1doma Secreting Mcab To Pefloxacinum And Immunological Identification

The objective aimed at preparing the hybridoma of PEF and applied to explore itsimmunological characteristics for immunological rapid determination of PEF residues.Theresults showed that:1. Basis on analysis of molecular structure and immunogenicity of PEF,immungen andcovering antigen were prepared by EDC coupling PEF to BSA or OVA.By the ultravioletabsorb spectral curve and SDS~PAGE electrophoresis,get the conjugate ratio. It turned outthat the PEF-BSA and PEF-OVA were synthesized successfully,and the molecule conjugateration of PEF to BSA and OVA were8:1and6:1respectively.2. To immunize BALB/c mice using PEF-BSA,the antibody was identified byCi-ELISA,in order to prove that the PEF-OVA as a bag was antigen is feasible,we choose theCi-ELISA method.The result was the five mice immune serum antibody titer all reached1:4000by Ci-ELISA,the fifth mice was arived1:32000.Ci-ELISA test positive serum and resultsshowed that feasible.3. BALB/c mice was chosen from five mice BALB/c immunized with PEF-BSA forcell fusion,the result of the positive by Ci-ELISA for the hybridoma.Scanning the positive cellwith the(limiting dilution analysis)LDA for the ascites.The result was that scanning twohybridoma lines of5E2and4D8,the cell fusion ration were31.18%and45.65%respectively,the ration of objective were100%.4. Caryotype analysis of hybridoma of PEF mAb with the method of colchicine;comparison of ELISA titer of ascites and purified ascites;identify and classify the antibodysubtypes using the protein kit, assay the the stability of secretory antibodies in the indirectELISA.The results showed that the isotpe of them was IgG1, caryotype analysis ofhybridoma of PEF mAb was about90,comparison of ELISA titer of ascites and purifiedascites,the titer was higher;the anabiosis cells was indentified by Ci-ELISA,prove that hasgood stability. In this study,we have successfully screened out of the cell line, which could secretePefloxacin antibody.Finally, we confirmed that the cell lines are fusioned by the cells ofimmune spleen and SP2/0through subtype analysis, chromosomal counts and identification ofstability, it has provided the basis for the detection of pefloxacin residue.