The Exploratory Study On Simultaneous Screening To The Residues Of Three Varied Veterinary Drugs

Veterinary drug resides has been a focus problem in food safety field, which can give rise to direct and indirect harm for body, such as propagated and embryonic toxicity, nerve toxicity, immunity reduction, hypersensitive response , carcinogenesis , malformation , mutation and antimicrobial resistance,ect. At present, the detection method of resides mainly includes biological effect determination , physico-chemical determination, HPLC, mass spectrometry , GC-MS, immunological determination , biosensor determination , ect. However , whether traditional conventional determination or standard accurate determination all universally exist some shortcomings, such as determined target being single, operating steps being miscellaneous, cost and instrument being expensive. Especially when simultaneously detecting various unknown pollutants, the shortcomings of lots of workload and time-consuming or force-dissipating ect appear undoubtedly. The detection technology need of fast, sensitive and accurately screening multiple unknown pollutants once also is increasing steadily. For this reason, the research develops the study for the immunological determination of CAP and SM2 from small molecular veterinary drug CAP,SM2 and CPFX. Meanwhile the research also explores the application prospect of single FV and fusion single FV in the broad-spectrum detection of residual veterinary drugs and provides a reliable method base for the fast detection of the livestock and poultry export-import in china.The preparation of CAP and SM₂ complete antigen CAP and SM₂ were coupled with BSA and OVA by using diazotization to be immunogen and detecting antigen. With Agarose Gel Electrophoresis and Polyacrylamide Gel Electrophoresis method, the coupling effect of coupling product was analyzed and the molecule coupling ratio of the CAP and BSA, OVA being 28:1 and 21:1 and Protein concentrations separately being 3.16mg/mLå’Œ2.28mg/mL, meanwhile the molecule coupling ratio of the SM₂ and BSA ,OVA being 23:1 and 17:1 and Protein concentrations separately being 3.24mg/mLå’Œ2.29mg/mL were calculated by using indirect competitive ELISA. The electrophoretic result showed that small molecular has been coupled to vector protein and successfully constructed the immunogen and detecting antigen of two drugs.The preparation and characteristic study of CAP and SM₂ monoclonal antibody The female BALB/C mice aged 8 weeks were separately immunized by CAP-BSA and SM₂-BSA prepared. Conventional cell fusion technique makes spleen cell fused with cell line SP2/0, which positive clone was examined by indirect ELISA. After 3 clones , respectively can screen 1 strain hybridona cell 4B12 which can stably sret anti-CAP antibody and 2 strain hybridona cell 2G2 , 3D7 which can stably sret anti-SM₂ antibody ( among them, the high titer 3D of anti-SM₂ antibody identifies its specificity). The chromosome average number of hybridona cell was 50±2 and sreting antibody all belonged to the subclass of IgG1 and the relative molecular weight of purifying antibody respectively was 157920,162280 and the affinity constant was respectively 8.26×10⁸M^-1,6.09×10⁸ M^-1 and the infective titer was respectively 2.56×10⁵ and 1.28×10⁵, which the result of cross-reaction was good. Using ascites method can produced great quantity monoclonal antibodies and the ascites was purified by CA-AS. The results showed that the recovery of CA-AS was separately 55.12% and 59.23% for IgG and the rate of purification was separately 80.47% and 84.25%. The experiment also showed that the method of purifying mouse ascitesfluid IgG is simple operation, low cost and better effect of purification and recovery.The establish of CAP and SM₂ competitive inhibitory ELISA testing method Using CAP Monoclonal Antibody respectively constructs two testing methods of one-step competitive inhibitory ELISA( the enzyme labeling of monoclonal antibody) and two-step competitive inhibitory ELISA(commercial enzyme-labeled sond antibody).The regression equation and correlation coefficient of two testing method for CAP:y=-25.411x+97.041,R2=0.9889å’Œy=-27.768x+103.91,R2=0.9859. The linear range was separately 0.93²50nμg/L and 1.87⁷50ng/L and The minimum detection limit for CAP was respectively 3.5ng/L and 9.7ng/L。. The chicken simulated sample was tested by two ELISA method constructed, which recovery of testing sample was separately 98.607% and 85.152%; The regression equation and correlation coefficient of two testing method for SM₂ :: y=-26.728x+100.54 , R2=0.9958å’Œy=-28.049x+105.7. The linear range was separately 0.67²10ng/L and 1.53⁶20ng/L and The minimum detection limit for CAP was respectively 4.9ng/L and 5.4ng/L. The chicken simulated sample was tested by two ELISA method constructed, which recovery of testing sample was separately 99.331 % and 88.687%. The synthesis and expression of CAP, CPFX and SM₂ single chain variable fragment (scFv) antibody The heavy chain and light chain variable gene of CAP, SM₂ and CPFX monoclonal antibody was amplified by RT-PCR method from the total RNA of CAP 4B12, SM₂ 3D7 and CPFX 3C6 hybridona cell strain. Single chain variable fragment gene (VH-Linker-VL)was assembled by SOE-PCR method, which was cloned with pMD18-T vector. The sequence analysis showed that CAP-ScFv was 714 bp encoded a deduced amino acid sequence of 238 residues, CPFX-ScFv being 729 bp encoded a deduced amino acid sequence of 242 residues, SM₂-ScFv being 774bp encoded a deduced amino acid sequence of 258 residues, which 15 amino acids all were the flexible Linker connection between heavy chain and light chain. Gene sequence was analyzed by Igblast to get the variable gene of antibody light chain and heavy chain, which was in agreement with the characteristics of the mouse antibody variable region. Analysis of Amino Acids homology had the typical domains (IGV )of immunoglobulin and had high homology with single chain antibody registered, which was the mouse antibody variable gene sequence of the functional rearrangement.The cloning , expression and activity assay of pGEX- CAP-CPFX-SM₂ fusion Single Chain Fv Antibodies pGEX-CAP-CPFX-SM₂ Single Chain Fv Antibodies was connected in gene level and constructed prokaryotic expression vector, which was inducted by IPTG. The molecular weight of expression fusion protein banded GST marker was determined as about 100 ku. ELISA detection showed that the expression fusion protein had good immunological reactivity. To sum up, the monoclonal antibodies of the small molecular veterinary drugs CAP and SM₂ were separately prepared n the research. After constructing the direct ELISA and indirect competitive inhibitory ELISA of two drugs fast, sensitive and efficient, convenient, colloidal gold strip test was carried up. Meanwhile the variable gene (V_H,V_L) of CAP,SM₂ and CPFX monoclonal antibody were cloned and SCFv gene (V_H-Linker-V_L) was assembled. After the S_CFv connected gene of three drugs being expressed by using pGEX-6P vector and being purified by affinity chromatography, the reactivity of the fusion expression protein tested by indirect ELISA method is good for three drugs, which can provide experimental basis for establishing wide-spectrum single chain antibody and screening the quick determination method of three kinds of common residual drugs at least. The research also broke an immunology examination new path of quickly screening small molecular drugs.