Preparation And Application Of The Monoclonal Antibodies Against Sulfamethoxazole And Development Of Colloidal Gold Immunochromatographic Assay For Repaid Detection Of Sulfamethoxazole Residues

In the veterinary clinic,Sulfamethoxazole(SMZ) is mainly used to treat respiratory and urinary tract infection.It has wide antibiotic spectrum,formal curative effect,stable property,ease to use inexpensive,and many other advantages. Food derived from animals treated with Sulfamethoxazole may be contaminated with this drugs will influence human health because of abuse of Sulfamethoxazole. Thereby,we need establish a series of effective methods to meet different conditions of animal SMZ residues detection in food.At present,the main method used for sulfonamides detection include microbiological method,chromatography,immunology analysis,etc. As a rapid,specific and sensitive method,to find and establish the best way for detecting Sulfamethoxazole residues.In this research,monoclonal antibodies with high sensitivity specificity and valence were prepared through monocolonal antibody technology,the ELISA detection method was founded, and test strips of immunochromatographicy are developed.1.Synthesis of SMZ complete antigens.Sulfamethoxazole is a half antigen.In order to become a complete antigen,a carrier is necessary.SMZ-BSA,SMZ-OVA and SMZ-HSA were prepared by N-(3-Dimethy-laminoproyl)-N'-ethylcadbodiimide-hydrochloride (EDC),Glutaraldehyde and Diazotation reaction method respectively.After purifid,UV-spectrum technology is used for confirming that Sulfamethoxazole has been conjugated to BSA,OVA or HSA,which have changed their ultraviolet absorption and shapes.The hapten-protein conjugate rate was 23.3:1(SMZ-BSA),17.7:1(SMZ-OVA),24.5:1(SMZ-HSA) seperately.2.Preparation of monoclonal antibodies againt SMZ. One mouse(BALB/C),7 weeks old,was immunized subcontaneously with SMZ-BSA.The positive cells were sub-cloned by limiting dilutions three times.This procedure resulted in a stable clones: 1C5. The anti-drug Sulfamethoxazole ascites monoclonal antibody was got. Hybridoma 1C5 producing anti-SMZ monoclonal antibody(McAb) was used to inocluate into BALB/C mice abdominal cavity.The results showed that the concentration of McAb was 24.9mg/mL,the titer of the McAb was 1:80000.Indirect competitive ELISA (CiELISA) method was established for 1C5 ascites, there is a standard curve,the curve equation and the repression was y=0.3068x+0.0824 (R2=0.9955), the 50% inhibition concentration was 22.9ng/ml,the limit of detection of this assay was established to be 1.02ng/mL,and the limit of determination was 5.39ng/mL. To test the speciality of antibody,7 Sulfonamides were used in the competitive ELISA as the competitive antigen.The result proved that the ascites shows no cross-reaction to other 7 Sulfonamides.3.Development of SMZ colloidal gold immunochromatographic strip. Tri-sodium citrate with aqueous gold chloride to make 25nm colloidal gold which was coupled with the monoclonal antibodies after identification.The conjugation solidified to glass fibers of Ahlstrom 8964,SMZ-OVA was immobilised on nitrocellose membrame (NC) of Millipore 135 to prepare for strips. The various factors and conditions of the immuno-chromatographic lateral-flow assay were explored, and the optimal reaction conditions of the assay were ascertained. A GICA for rapid detection of Sulfamethoxazole residues in food derived from animals was successfully established. The GICA device had a cut-off value of 100ng/ml for detecting the SMZ dissolved in the PBS. The cross-reactive rates of GICA to other 6 antibiotics were negligible.4.SMZ addtive reclamation test. Additive reclamation test had been done for animal foods which with relatively more SMZ used. The measured drug additive reclamation test rates were:milk 92.4% to 112.6%,the whole eggs 86.9% to 128.2%,chichen 68.4% to 84.5%. And samples were treated with the test strip to verify that the accuracy rate of milk detection was 91.7%, the accuracy rate of eggs detection was 87.5%, the accuracy rate of chichen detection was 83.3%.The results detected by the strip show that the strip can meet the testing requirements.Experiment results showed that the ELISA and GICA method established in animal food SMZ residue detection was convenient, fast and accurate for rapid detection. The method of detection for SMZ could be used for reference in the development of the detection of other veterinary drugs.