The Establishment Of Enzyme Enhancement Chemiluminescence Immunoassay For Sulfachlorpyrazine Sodium

Sulfachlorpyrazine sodium(SPZ)is a kind of the sulfonamides(SAs), which is usually used to therapy coccidia in chicken.While the residues of SPZ in edible tissues may occur because of the abusing or insufficient withdrawal time, and this could cause drug resistance problem to humans. The immunological methods are usually used in screening drug residues in animal tissues with its high sensitivity and specificity. However, besides some papers have reported about enzyme-linked immunosorbent assay(ELISA) methods, there are few reports about Enzyme Enhancement Chemiluminescence immune analysis(CLIA). In this study the monoclonal antibody against SPZ was prepared, then the CLIA method was established on the basis of the antibody. Otherwise, the sructure TS was synthesized and the polyclonal antibody was prepared in order to establish rapid detection methods of residues of SAs in edible animal tissues.1.The preparation and identification of SPZ monoclonal antibody. The SPZ was conjugated with ovalbumin(OVA) for preparation of immune antigen through diazotization method. After immunization,the mouse with higher titer was sacrificed and the spleen cells were fused with myeloma cells SP2/0.The growing hybridoma cells were screened more than once, then the positive hybridomas were subcloned by limited dilution method for three times. Then monoclonal antibodies(Mab) were produced in mouse ascites and puri?ed. The results ELISA showed that the titer of Mab D9 against SPZ was 1:4×105 and the half inhibition concentration(IC50) was 326 ng/m L. In addition, the specificity of the MAb D9, estimated as the cross-reactivity values(CR) with other experiment drugs, was below 5%.The results indicated that the Mab performed well in specificity.2. CLIA method for SPZ. On the basis of Mab D9, CLIA method for SPZ residue was established.The sensitivity of the developed CLIA was estimated as the IC50 value(151 ng/m L) with a linear working range between 0.02 and 4 μg/m L, and the regression equation is y=-0.3602x+1.2874(R2=0.981). The limit detection of SPZ in samples was 36.3ng/g. The recoveries of SPZ from chicken muscle forti?ed at levels of 75-85%. The relative standard deviations of intra and interbatch were 7.02%and 8.5%.3. Preparation and identification of polyclonal antibodies against sulfonamides. Firstly, TS was synthesized and then conjugated with BSA for preparation of immune antigen through1-ethyl- 3-(3-dimethyl aminopropyl) carbodiimide hydrochloride(EDC) method. After immunization, the anti-serum titer was determined with ELISA.The results showed that the titers of the two mice were 1:2.56×105 and1:1.28×105. The better one was selected for the following experiments. The anti-serum was tested in ci-ELISA and CLIA for competition with 8 sulfonamides, and the antibody showed the best sensitivity towards TS with the IC50 values were 33ng/m L and 7.9ng/m L. The cross-reactivities of the sulfonamides with five-membered heterocycle were above 26%, and the sulfonamides with sixmembered heterocycle showed less competition(CR<11%). That means the antibody could recognize some SAs with different R groups.