Study On The Residues Of Sulfonamides By Enzyme Immunoassay

Sulfonamide are synthetic bacteriostatic drug,widely used in animal husbandry and as feed additives.As a result,food derived from animals treated with sulfonamides may be contaminated with those drugs,and will influence human health, cause allergic reactions, and could result in resistance in pathogeic organisms because of the extensive use of sulfonamides.To safeguard the public heath,many countrys and organisations have established the Maximum Residues Limits(MRLS) of 0.1ug/g or even none.At present,the primary methods for detecting Sulfonamides Residues, high performance liquid chromatography(HPLC),gas chromatography(GC) and mass chromatography, are not suitable for screening a large number of samples.for they are time-consuming,costly and needing high skill.Enzyme-linked immunosorbent assy(ELISA) as a rapid,special and sensitive method has been gradually applied to veterinary drug determine. Immunoassay Screening methods for sulfonanides in foods and related materials have been reported in the literature.But the research on the multi-residues in veterinary drug is just beginning,and there are many question eager to solve both in theory and practice.This research synthesized two haptens H1,H2 which contain the common structure of Sulfonamide,by chemical method and developed anti-sulfonamides antibody. Anti-sulfamethoxazole antibody has been developed and the ELISA method has established for detecting SMZ ,and the method has been applied to screen SMZ residues in raw milk. The two haptens was synthesized through a series of chemical reactions of ASC and PABA.The products were successfully identified by UV scan, IR and MS .Through the EDC,DCC and MA methods,we prepared the immune antigen and coating antigen.UV-scaning identified the conjugates.The ratio of haptens to carrier protein is 10.3:1 and 9.6:lfor H1-HSA and H1-BSA.Six rabbits were injected with the conjugates and anti-sulfonamides antibodies were developed.All of the titer of antiserum were above 1:100,000 by ELISA assay. Meanwhile,the limit of detection(LOD)of this assay was calculated to be 0.065,0.046,0.045,0.032,0.015 g/ml for SQ, SMM , SMD , SMZ , SC all of which were under the MRLS 0.1 ug/ml .However, the ELISA assay was not sensitive to SD, SM2 whose LOD are 0.86,1.0 g/ml.The antibody can be used to detect several sulfonamide drug residues .We prepared Enzyme labelled hapten and establised competitive direct ELISA.Those study showed the difference in the N1 position' s energy conformation and electrical density and property which were the determinate factors of Antibody combining with sulfonamides.We established the immune method for detecting SMZ. SMZ was bounded to BSA or OVA using glutaraldehyde as the complete antigen, and developed monoclonal antibody and antiserum which is special and sensitive to SMZ.The competitive indirect ELISA method for screen SMZ residues was established with thisantiserum.Calibration graphes were prepared by plotting Ig drug concentration against percentage inhibition.The limit of detection of SMZ was 8.74ng/ml,and the limit of quantitative (LOQ)was 71.46ng/ml in PBS.The cross reaction with other six sulfonamides was low than 0.1% , which proved the antibody was very special.The standard curve of CiELISA was also established in raw milk condition and the curve indicated that the lower detection limit was 6.14ng/ml,the LOQ was 25.4ng/ml and the IC50 was 398ng/ml.The recovery ratio was 71.6%-106%,while SD was 4.5%-10.27%.The sensitivity and accuracy of the competitive enzyme immunoassay seemed to be satisfactory for quantitation of SMZ in milk,and fulfiled the demands of the MRLs regulation.