| Sulphonamides have a broad spectrum of antibacterial activity and are frequently added into feeds for the prevention or treatments of vary animal diseases. However, this result in the residues in edible tissues, and finally do harm to food hygiene and human health.Some countries have established total sulphonamides tolerance limit in tissues for human consumption at 0.1mg/kg.The international has established many chemical and physics methods for detecting sulfonamides. Though some of these methods are sensitive and accurate, they are not suitable for screening a large number of samples because these methods are time-consumption, and costly and highly skilled. Enzyme-linked immunosorbent assay (ELISA) as a rapid, special and sensitive biological method is gradually applied in drug residues assay. This research synthesized the antigen and developed the antibodies, and established the ELISA method for detecting SMD. In the experimental condition, the residues of SMD in pig and chock's tissues were determined.To prepare monoclonal antibodies (mAbs) against SMD, SMD was bound to BSA or OVA using glutaraldehyde as the coupling reagent, and using UV spectrophotometer scanning identified the conjugates. SMD was linked to a carrier protein via the N4-position. In doing so, N'-substituents, which are specific for individual sulfonamides were readily exposed to the immune system and this resulted in the production of antibodies that were highly specific for the particular sulfonamide drug used as the hapten.Four mice (BALB/c), 7 weeks old, were immunized subcutaneously (sc) with 50μg of SMD-BSA conjugates in 100μl of PBS emulsified with 100μ1 of complete Freund's adjuvant. Booster injections (sc) with 25Hg of the immunogen in 100μl of PBS emulsified with 100μl of incomplete Freund's adjuvant were given at 2~3 week intervals. After three booster injections, the mice were primed with a final booster injection (intraperitoneally). Three days later, spleen cells were isolated. The fresh spleen cell was used for fusion with myeloma cell. Using iELISA and icELISA to screen the positive cell. The supernatant of the positive well reacted to the SMD-OVA and 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.Six mice (BALB/c) were injected intraperitoneally with 3E8 cell to produce ascites.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 absorbance obtained in the absence (Bo) and the presence (B) of SMD in standards and samples as follows: binding= B/Bo-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, eight sulfonamides were used in the competitive ELISA as the competitive antigen. The result 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 were determined by the method. The results show that withdraw! period of SMD in chook is beyond fourteen days.
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