| Sulfonamides (SAs) are synthetic bacteriostatic drugs which are widely used in animal husbandry and as feed additives. Also, in internal and international, the sulfonamides residues detections in food derived from animals was one of the important items in the food hygiene standard. As a rapid, special and sensitive method, Enzyme-linked immunosorbent assay(ELISA) has been gradually applied to veterinary drug determination.Presently, the immunodetection for single Sulfonamide has been greatly progressed, and it will be more practical if the detection of sulfonamides multi-residues could come true. But it is difficult because sulfonamides have various categories and complicated structures. To carry out the sulfonamides multi-residues detection, first of all, we have to develop group specificity antibodies, and the key point of that is to synthesize an artificial hapten which has the common structure of sulfonamides.According to the substituent group at benzenesulfonamido terminus, Sulfonamides can be classified into the groups of five-membered and six-membered heterocycle. Up to now, no one has studied the multi-residues detection of sulfonamides with five or six-membered heterocycle. This research have synthesized artificial hapten TS which has the common structure of the Sulfonamides with five-membered heterocycle, and developed anti-sulfonamides antibody that can recognize 4 sulfonamides. Also, the ELISA methods has been established for detecting sulfonamides, and applied to detect multi-residues of sulfonamide in chicken muscle. Meanwhile, we have discussed the basic rules of the sulfonamide multi-residues by immunoassay.After two-step chemical reactions, a sulfathiazole derivative named TS was synthesized by ASC and ATAA. TS was proved to be our expected production by mass spectrogram detection. The complete antigen, TS-HSA and TS-BSA, were prepared by EDC method. UV-scanning identified the conjugates. The ratio of haptens to carrier protein is 44:1 and 21:1 for TS-HSA and TS-BSA.Mice (BALB/c) were immunized by TS-HSA, in the way of intrasplenic and routine method. Compared to the routine way, intrasplenic immunization methods could save immune-time, but the title of antiserum was lower, and the avidity and specificity of antiserum were not as good as the routine way. Therefore, we isolated the mice spleen cells which were immuned by routine way, to fusion with myeloma cells. The positive cells were sub-cloned by limiting dilutions three times. Finally a stable clone 3D3 was found and then expanded cultured. Also, ascites were produced by injecting 3D3 cells to mice intraperitoneally. The suitable concentration of coated antigen and ascites titer were determined by chessboard test, and the ascites titer reached 1:2,560,000, the reaction concentration was 1:160,000. The cross-action of ascites to other antibiotics was negligible.Indirect competitive ELISA was established with this ascites. Calibration graphs were prepared. The limit of detection of this assay was calculated to be 14.4,6.3,18.2,33.88 and 4.63 ng/mL for ST,PST,SST,SMZ and 4 mixed drug(ST,PST,SST,SMZ), all of which were under the MRLs 0.1μg/mL. Also the correlation value is low. So the ELISA can be used in practical detection.The CiELISA method has been applied to detect multi-residues of sulfonamide in chicken muscle. Intervention disappeared when the extract were diluted 8 times. The recovery ratio were between 72.9%～100% when adding 100,1000 and 5000ng/mL 4 sulfonamides mixture(ST,PST,SST,SMZ) to chicken muscle. The intraassay coefficient of variation were between 2.56%～6.63%, and the interassay coefficient of variation were between 5.31%～9.72%. The reproducibility was fine.
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