Preparation Of Anti-Sulfonamides Antibody And Study On Immunoassay Specific For Sulfonamides

The sulfonamides are an important class of broad-spectrum antibacterial drugs which are widely applied in the treatment and prophylaxis of bacterial infections and commonly fed to food animals for growth-promoting purposes as feed additives. However, the residues of sulfonamides occur in edible animal food and present a potential danger to human health because of improper use of these drugs. In order to establish immunological methods with high sensitivity to screen the residues of these sulfonamides three kinds of antibodies specific for sulfonamides were prepared and their specificity and application were studied. The main research and results contain the following parts.1. A monoclonal antibody (McAb) specific for sulfamethoxazole (SMX) was produced by hybridoma technology. The specificity of McAb was characterized by ELISA, SDS-PAGE. The results showed that SMX-McAb was of the IgG₁ type and its molecular weight was 162 000 daltons. The affinity constant of McAb with coated complete antigen was 2.3×10⁸ L/mol. There is a tolerance to three kinds of organic solvent for monoclonal antibody. The concentrations of SMX were linear from 10pg/ml 10ng/ml. The linear fit equation was: Y = 8.91+19.67857X and the sensitivity of the assay in terms of the inhibition rate of 50% for SMX is 0.82ng/ml. There was no cross-reaction between McAb and SMX analogs. The recoveries of SMX in milk was above 90%.2. A polyclonal antibody (PcAb) specific for SMX was produced by immunized New Zealand white rabbits with a synthesized antigen and the specificity of the polyclonal antibody was then characterized by ELISA. The result showed that the polyclonal antibody was specific for SMX. In order to select the detection method for using the kit, three ELISA methods were compared. These included HRP labeled antigen competition ELISA method, the HRP labeled antibody inhibition ELISA method and the sandwich-ELISA method. After considering the precision, sensitivity, accuracy and rapidness, the HRP labeled PcAb sandwich-ELISA method was selected as the detection method for the kit. Finally, the detection conditions were developed, and the precision, sensitivity and accuracy of the ELISA kit were evaluated in this experiment. The results showed that the sensitivity limits of the kit reached 20.91ng/ml, and at the range of 100μg/ml¹ng/ml, the values resulted in a good linear correlation with the contents of SMX present in the sample. The coefficient of variation (CV) for intra and inter-plate were 2.51% and 4.97% respectively. Overall, the results from the studies demonstrated that the kit may be used for quantitative determination of SMX in edible animal food.3. A polyclonal antibody (PcAb) specific for sulfadimethoxine (SDM) was produced by immunized New Zealand white with a synthesized antigen and the specificity of the polyclonal antibody was investigated by ELISA. The results showed that the polyclonal antibody was specific for SDM. ThenPcAb was conjugated to thermo-sensitive hydrogel poly-N-isopropylacrylamide (pNIPA) to form antibody bound hydrogel (pNIPA-Ab_SDM). The complete antigen was labeled by the fluorescein isothiocyanate (FITC) to form a fluorescence antigen complex (FITC-Ag_SDM). The precipitation immunoassay was established based on the competitive binding of free SDM and fluoresceinated antigen (FITC-Ag_SDM) with limited amount of pNIPA-Ab_SDM. The results showed that the lineal range of SDM detection was 1ng/ml¹00μg/ml and the detection limit was 0.64 μg/ml. The recoveries of SDM in milk was above 90%. The result showed it was used in the residue detection of SDM in sample.4. A polyclonal antibody (PcAb) specific for Sulfanailmide (SN) was produced by immunized New Zealand white rabbits with a synthesized antigen and the specificity of the polyclonal antibody was then characterized by EL1SA. The results showed that the polyclonal antibody was specific for SN. PcAb was conjugated to thermo-sensitive hydrogel poly-N-isopropylacrylamide (pNIPA) to form antibody bound hydrogel (pNIPA-Ab_SN). The complete antigen...