有机磷农药多残留酶联免疫吸附分析方法的研究

上海交通大学硕士学位论文有机磷农药多残留酶联免疫吸附分析方法的研究姓名：张敏申请学位级别：硕士专业：农药学指导教师：陆贻通20081201I(ELISA)ELISA1.O,O-4--N-(3-)DENP2.DENPBSAOVADENP-BSADENP-OVA1211713.DENP-BSA11024001256004.9DENPIIDENPDENP5.ELISA0.0037mg/L0.033mg/L108.00%~112.00%IIISTUDYOFMULTI-RESIDUEELISAFORORGANOPHOSPHORUSPESTICIDESABSTRACTTheaimoftheresearchisdevelopingmulti-residueenzymelinkedimmunosorbantassay(ELISA)fororganophosphoruspesticides(OPs).GenerichaptenforOPswassynthesizedandconjugatedwithproteintoproducegenericimmunogenwhichwastheninjectedintorabbitstogenerategenericantiserum.Withtheantiserum,ELISAmethodforseveralOPswasthenestablishedandcouldbeappliedformulti-residuedetectionofOPs.Mainresultsarelistedbelow:1.GenericHaptenO,O-diethyl-N-(3-carboxypropyl)phosphoramidothioate(namedDENP)wassynthesizedfromthereactionbetweenO,O-diethylthiophosphorylchlorideand4-aminobutyricacid.2.Genericimmungen(DENP-BSA)andcoatingantigen(DENP-OVA)weresynthesizedbyconjugatinggenerichaptenDENPwithbovineserumalbumin(BSA)andovalbumin(OVA),respectively.Conjugationratioswere12:1and17:1respectively.3.GenericantiserumwasobtainedbyimmunizingtworabbitswithDENP-BSA.Titersofantiserawere1102400and25600.4.Affinitybetweenantiserumand9OPswasstudied.Theresultsshowedthat:OPsofnearlythesamestructurewithhaptenDENP,includingphorate,demetonSanddisulfoton,hadhighestaffinitywithantiserum;O,O-dimethylphosphorothioatepesticidesincludingomethoateanddimethoatewerelessaffinitivewithantiserum,thoughtheyalsohadlinearhydrocarbonstructurelikeDENP;OPscontainaromaticorheterocyclicringincludingphoxim,quinalphos,diazinonandchlorpyrifoshadleastaffinitywithantiserum,eventheyhavethesameO,O-diethylphosphorothioatewithDENP.5.Multi-residueELISAforphorateanddemetonSwasdevelopedwiththeLODof0.0037mg/Land0.033mg/L,respectively.AndtheIVrecoveriesofphorateanddemetonSinthewatersampleswerefrom108.0%to112.0%,KEYWORDS:organophosphoruspesticides,multi-residue,generichapten,genericimmunogen,genericantiserum,ELISAVAbAntibodyAgAntugenBSABovinSerumAlburminCVTheCoefficientofVariationsDCCNN’-DicyclohexylcarbodiimideDENPO,O-Diethyl-N-(3-Carboxypropyl)PhosphoramidothioateO,O--N-(3-)DMFN,N-DimethylformamideNN-EDC1-3-DimethylAminopropyl-3-EthylcarbodiimideHydrochloride1--3-ELISAEnzymeLinkedImmunosorbantAssaysIAImmunoassayNHSN-HydroxysuccinimideN-ODOpticalDensityOPsOrganophosphorusPesticidesOVAOvalbuminChickenPBSPhosphateBufferSaltSDStandardDeviationTMB3,3’,5,5’-Tetramethylbenzidine2009225□□√“√”2009225200922511.1[1·2][3][4]1.2-[5]2(EnzymeLinkedImmunosorbantassayELISA)[6]1.3[7]1.3.1(MW2500)-A.[8]B.3~6C[9]C.3D.A.(FH),(EDC)[10]B.()[11]()[12]C.[20]D.MP1MP2[13]NanjuLee[14]AntonioAbad[15][16]E.O,OO(),(BSA),[17]milbemycinoxime[18][19]1.3.24[20~23]1.3.3PolyclonalantibodyPcAbBB[2425](MonoclonalAtibody,McAb)BBBBB[2425](SinglechainFvScFv)FabScFv5FabFabELISA1.3.4ELISA1.3.4.1.ELISA96[2627]ELISAhorseradishperoxidase,HRPo-phenylenediamine,OPD3,3’,5,5’-tetramethylbenzidine,TMB1.3.4.2.ELISAELISAELISA6ELISAELISAELISAELISAdirectcompetitiveELISAELISAindirectcompetitiveELISA[26](1)ELISA(2).ELISAIgGELISAELISAELISAELISA7ELISA1.3.4.3.ELISAELISAI=(A-D)/[1+(x/C)B]+D(1)ABC50%D[28]1-1S-XYLODLOQIC5050%Fig.1-1Thestandards-curveofDose-ResponseTheX-axisistheconcentrationofanalyte(LogC).TheY-axisistheabsorbance.LODisthelimitofdetection.LOQisthelimitofquantification.IC50ismiddleofassay,concentrationofanalytethatcause50%inhibition.1BADyDXC−=++…………………………………………………………………1011BByDBADxC−==−+……………………………………………………………28lgxy1-S-sigmoidalcurve1ELISAB/B0%2ylgxB/B0%2-Slgxy-SB/B0%-SB/B0=50%IC50IC5050%10%-3×SD20%~80%5050()100%()ICCRIC=×1.4[5]genericantibodiesJ.N.Banks[29]O,O-ELISA9IC504.88.236.291.1g/mL.Jeffre.C.Johnson[30]O,O-O,O-4-(4-)-O,O-O,O--O-(-(4-))2KLHELISA0.1ng/mLO,O-1-2.O,O-,O-(-(4-))Fig.1-2SynthesisofO,O-Diethyl,O-[p-(4-Carboxybutyl)phenyl]PhosphorothionateM.J.C.Alcocer[31]-4--2-TPB3TPBKLHELISA0.30.6ng/mL252(CHO)PO2OCHCHCHCOOH1-3.-4--2-Fig.1-3Diethyltrans-4-phosphono-2-butenicacid10MiSoonJang[32]3--4-4KLHELISACl,-CH3,-NO2,-Br0.01g/mL1-4.O,O--O-(5--2)Fig.1-4SynthesisofO,O-Diethyl-O-(5-carboxy-2-fluorophenyl)phosphorothioate1.5PCRELISAELISAELISA111.61234ELISA121.7ELISA132.1[33]2.2.2.2.1O,O-99%EDC·HClBioBasicInc.,Canada4-N-NN-IgG-HRP20BSAOVA0.05mol/LpH=9.65%0.2mol/LpH=7.40.2mol/LpH=7.40.05%200.02mol/LpH=7.0(TMB)25mL3.3µL30%H2O2400µL140.6%TMBpH=5.52mol/LH2SO42.2.2UV-2410PCRE-52SZ-93JB-3YXQ-LS-100ASHB-MULTIFUGE3S-RCHRISTChristREVCOMultiskanAscentlabsystemsWellwashAClabsystems96CostarlabsystemsBS-210SIMSHHH.CP-T2.32.3.1(1)O,O-O,O-4-O,O--N-(3-)DENP152-1CH3CH2OCH3CH2OPSCl+NH2COOHOH-0CH3CH2OCH3CH2OPSNHCOOH2-1DENPFig.2-1SynthesispathwayofgenerichaptenDENPforOrganophosphoruspesticides(2)O,O-[34]1.0g5.3mmolO,O-5mL4-0.67g6.5mmolKOH0.69g12.3mmol5mLO,O-2h20mL5%NaHCO310mLpH=310mL0.53gDENP39.2%2.3.2DENPGC-MSDENPCDCl31H-NMR2.3.3O,O-DENP[35]12-216NHSEDCBSANH2CONHBSAorOVAOVAorCH3CH2OCH3CH2OPSNHCOOHClHNHNNH(CH3)2+CH3CH3CH2OCH3CH2OPSNHCOOCH3CH2OCH3CH2OPSNHCl2-2Fig.2-2SynthesisofgenericimmunogenandcoatingantigenforOPs252mgDENP0.2mmol3mLDMF46mgNHS0.4mmol78mgEDC0.4mmol10133mgBSA2µmol15mL0.2