Development And Application Of Enzyme-Linked Immunosorbent Assay Kit For Detection Of Sulfamethoxydiazine Residues

Sulphonamides have a broad spectrum of antibacterial activity and are frequentlyadded into feeds for the prevention or treatment of vary animal diseases. However, thisresult in the residues in edible tissues, and finally do harm to food hygiene and humanhealth. Many countries and international organizations have established totalsulphonamides tolerance limit in tissues for human consumption at 0.1mg/kg.Many chemical and physics methods have been established for detectingsulfonamides. Though some of these methods are sensitive and accurate, they are notsuitable for screening a large number of samples because these methods aretime-consumption, and costly and highly skilled. Enzyme-linked immunosorbent assay(ELISA) as a rapid, special and sensitive biological method is gradually applied indrug residues assay. This research synthesized the antigen and devoloped theantibodies, and established the ELISA method for detecting SMD. In theexperimental condition, the residues of SMD in pig and chook's tissues weredetermined.To prepare monoclonal antibodies (mAbs) against SMD, SMD was bound to BSAor OVA using glutaraldehyde as the coupling reagent, and the conjugates wereidentified using UV spectrophotometer scanning. SMD was linked to a carder proteinvia the N⁴-position. In doing so, N¹-substituents, which are specific for individualsulfonamides were readily exposed to the immune system and this resulted in theproduction of antibodies that were highly specific for the particular sulfonamide drugused as the hapten.Four mice (BALB/c) were immunized subcutaneously (sc) with 50μg ofSMD-BSA conjugates in 100μl of PBS emulsified with 100μl of complete Freund'sadjuvant. Booster injections (sc) with 25μg of the immunogen in 100μl of PBSemulsified with 100μl of incomplete Freund's adjuvant were given at 2 to 3 weekintervals. After three booster injections, the mice were primed with a final boosterinjection (intraperitoneally). Three days later, spleen cells were isolated. The freshspleen cell was used for fusion with myeloma cell. Using iELISA and ciELISA toscreen the positive cell. The supernatant of the positive well reacted to the SMD-OVAand could be competed by SMD. The positive cells were cloned by limiting dilutions(three times). This procedure resulted in three stable clones: 3E8, 4A7, and 2D2.Indirect competitive ELISA (icELISA) was established with this ascites.Calibration graphs were prepared by plotting log(SMD concentration) againstpercentage binding. The percentage binding was calculated from the absorbanceobtained in the absence (B₀) and the presence (B) of SMD in standards and samples asfollows: binding= B/B₀.The limit of detection of this assay was established to be 1.766ng/ml, and the limit of determination was 11.274 ng/ml. To test the speciality of antibody, eightsulfonamides were used in the competitive ELISA as the competitive antigen. Theresult proved that the ascites shows no cross-reaction to the other eight sulfonamides.To validate the credibility of the method, Chook and pig were given some SMD.The sampers (liver, kidney, and serum) of chook and pig were collected, and weredetermined by the method. The results show that withdrawl period of SMD in chook isbeyond fourteen days.