Study On Elisa Based On Monoclonal Antibody For The Detection Of Quinolones And Sulfonamides

In this thesis two veterinary quinolones and sulfonamides monoclonal antibodies were prepared. And an ELISA for the detection of quinolones and sulfonamides was established.Norfloxacin connected with Bovine Serum Albumin, BSA, using NHS, which was used as immunogen. Mouse spleen cells were fused with myeloma cells, hybridoma cells were obtained by screening. By applying limited dilution, the cell lines, named4C6F6D, which could strain secreting antibodies were ultimately attained. Subtypes were dentified as: heavy chains, IgG3, light chain, Kappa. The amount of antibody, the dilution of enzyme labeled antigen, blocking buffer, pH and other conditions were optimized, and a direct competitive ELISA method was established. The half-maximum inhibition concentration (IC50) was0.45±0.07μg/L, the detection limit (IC15) was0.09±0.02μg/L. The sensitivity range of CIP, NAL, ENR, PEF, ENO, OFL, OXO, FLU, FLE, and LOM was0.27-8.54μg/L; the cross-reactivity rate was5.27-166.67%. The sensitivity range of MAR, SAR, DIF, GAT, PIP and SPA was14.21-88.13μg/L, the cross-reactivity rate was0.51-3.17%.Two kinds of sulfa artificial haptens were coupled to carrier protein KLH to obtain immunogen, which was applied to immunize mice. After a fustion of the preferred mouse spleen cells and SP2/0myeloma cells, the hybridoma was screened. A stable monoclonal antibody-secreting cell lines was ultimately pepared naming.4E7G4D. Subtypes were dentified as:heavy chains, Igf2a, light chain, Kappa. Based on the indirect competitive ELISA method we has optimized the heterologous and homologous package is original, the packaged amount, antibody dilution, blocking buffer, pH and other conditions. And we have established eight kinds of sulfomides standard curves. The sensitivity range of SQ, SCPA, SPMX, SMT, SME, SDDX, SMX and STZ was3.22-398.36μg/L. The cross-reactivity rate was0.81%-100%. In the direct competitive ELISA we also optimized the heterologous and homologous enzyme labeled antigen, antibody-coated amount, enzyme dilution, blocking buffer, pH and other conditions. And eight kinds of sulflomides standard curves were established. The sensitivity range of SQ, SCPA, SPMX, SMT, SME, SDDX, SMX and STZ was4.58-497.73μg/L; the cross-reactivity rate was0.92-100%.In this study we established enzyme-linked immunosorbent assays to detect quinolones and sulfonamides, which had high sensitivity and broad-spectrum. It was suitable for the rapid detection of multiple veterinary drugs residues in animal food.