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1998,21(2):6572JournalofNanjingAgriculturalUniversityB1ELISAa(,210095)B1(AFB1)2C322,,AFB1ELISAAFB1,B2,G1G2AFB1100%,314%,911%113%AFB112.5250pg,513%719%,AFB17910%8118%68,AFB11118%,AFB168217LgökgB1;ELISA;STS20714IndirectcompetitiveELISAforquantitativeanalysisofaflatoxinB1anditsapplicationChenLanmingandChenYongxuan(DeptofMicrobiology,NanjingAgricUniv,Nanjing210095)ABSTRACTHighaffinitypolyclonalantibodiesagainstaflatoxinB1(AFB1)wereobtainedfromrabbitsaf2terimmunizationofAFB12CMO2BSAconjugate.AsimpleandsensitiveindirectcompetitiveELISA(IDC2EILSA)forquantitativeanalysisofAFB1wasestablished.Thecross2reactivitiesofaflatoxinanalogswithAFB1antibodybyIDC2ELISAwere100%,3.4%,9.1%andlessthan1.3%forAFB1,B2,G1,andG2,respectively.TherangeofsensitivityinIDC2ELISAwas12.52250pg.Themeancoefficientsofvariationofwithin2bathandinter2batchwere5.3%and7.9%,respectively.AnalyticalrecoveriesofAFB1addedtothericeandmixedfeedswere79.0%and81.8%,respectively.11.8%ofthe68sampleswerepositiveforAFB1.ThecornsamplescollectedfromsomecountywerecontaminatedAFB1seriouslyatamaximumlev2elof682.7Lgökg.TheIDC2ELISAestablishedwasapplicabletodetectmoresamplesrapidly.KeywordsaflatoxinB1;ELISA;quantitativeanalysisapplicationB1(aflatoxinB1,AFB1)AspergillusflavusAapergillusparasiticus,AFB1,,(TLC)AFB1[1],a:1997210207,,,,,AFB1,AFB1AFB1ELISA(IDC2ELISA),AFB1111111111AFB12(AFB12C32carboxymethyloxime,AFB12CMO)Chu[2]20mgAFB120mL22(V()V()V()=141),40mg(carboxymethoxylaminehemihydrochiloride,Aldrich),5.5h,20mL,pH810,20mL3;,pH310,100mL4,,K362nmA,AFB12CMO11112AFB12CMO(BSA)Minh2Tan(water2solublecarbodiimide,WSC)[3]11113(AFB12CMO2HSA)112AFB111211:,2mL(300LgAFB12CMO2BSA1.5mL):4,2mL(250LgAFB12CMO2BSA1.5mL),4,(100LgAFB12CMO2BSA)11212IgG[4]113AFB1AFB1Truckss[5]:AFB1,10LgömL,,-20:1LgömLAFB1,,4,,0.05,0.1,0.25,0.5,1.0,2.5,5.0ngömLAFB1Chu[6]114AFB1IDC-ELISA:100LLAFB12HSA,373h4:0.01molöLPBST(pH7.4)3(ELISA46):50LL,50LL,371h100LL2HRP(14000),371h100LL,3710min50LL2molöLH2SO4A490nm6621115AFB1,A490nm,AFB1,,AFB1,(CAS20fx23600)BöB0,AFB12211AFB1-CMO-BSAAFB1(Mr:312),;1AFB12CMO2Fig.1SchematicdiagramforthepreparationofAFB12CMO2proteinconjugate1.AFB1;2.AFB12CMO2AFB1AFB12CMOFig.2IdentificationofAFB12CMObyTLCDevelopingsolvent:methanol2chloroform(1+9),AFB1C3,WSC(1)AFB12CMOK362nmA(E=20950,),AFB12CMO14172mg,7316%AFB1C3,(2)UV(3)212212114,1,3(R21R22R23)ELISA1100000,120000,120000(4)762:B1ELISA5ELISA5,4,,3AFB1AFB12CMOFig.3UVabsorbancespectraofAFB1andAFB12CMO4AFB1ELISAFig.4IndirctELISAtitrationofAFB1antisera2222Serumobtainedfromthesamerabbitsbeforeimmunization5AFB12BSAFig.5ProductionofantiseraagainstAFB12BSA6AFAFB1Fig.6Cross2reactivityofaflatoxinanalogswithAflaB1antibodybyIDC2ELISA212126AFAFB1IDC2ELISAAFB150%,AFB1,B2,G1G2:1126,36190,14139100ngömL;:100%,314%,911%113%AFB1AFB1,AFB1(1)21213IDC2ELISAAFB1IDC2ELISA614110-10molöL,AFB1AFB11156109Lömol86211AFB1-CMO-BSATable1SpecificityofantibodiesagainstAFB1-CMO-BSAAnalog33Cross2reactivity(%)Resultofthispaper[10]ResultsinthereferencesB1100.010010010010031003103B23.412517.710012.58202G19.1311.033.314.34206G21.311.01.31.18-43McAb;33DatafromELISA.213IDC-ELISA21311ELISAB0=1.0,,IDC2ELISAAFB1150000,(AFB12HSA)5LgömL7,0.255.0ngömL,LogitBöB0AFB1,r=-0.9925B022SD,6.4110-10molöL21312AFB10.15.0ngömL,513%719%(2)7AFB1IDC2ELISAFig.7Logit2logplotofAFB1standardcurvebyIDC2ELISA2IDC-ELISATable2Thevariabilityofwithin-bathandinter-bathofthestandardcurvebyIDC-ELISAAFB1Q(AFB1)ngmL()Mean(BöB0,%)SD(CV,%)Within2batch3Inter2batch5.014.41.08(7.6)13.72.43(17.8)2.526.92.81(10.4)31.32.00(6.5)1.051.12.13(4.2)52.54.71(8.9)0.583.71.71(2.0)82.45.23(6.3)0.2585.72.90(3.4)90.12.26(2.5)0.196.84.07(4.2)95.75.18(5.4)3Eachwasassayedin3wellson3differentmicrotiterplates21313ELISA,AFB1AFB1B1,B2,G1G2:100%,314%,911%113%2131410g(),AFB1962:B1ELISA,5400ngAFB1,,IDC2ELISA,33,AFB17910%8118%,7121615,1212%917%3AFB1Table3RecoveryofAFB1addedtoriceandmixedfeedasdeterminedbyIDC-ELISAAFB1AFB1AddedöLgkg-1()MeanSD(CV,%)Recovery(%)RiceMixedfeedRiceMixedfeedRiceMixedfeed4-2.30.12(5.2)-57.8-205019.75.63(28.6)47.97.78(16.2)98.695.85010045.73.60(7.9)76.40.00(0.0)91.376.410020074.64.05(5.4)132.213.29(10.1)74.566.1200400146.020.53(14.0)356.144.83(12.6)73.089.08AFB1ELISATLCFig.8CorrelationofELISAandTLCdatafordeterminationofAFB1incontami2natedsamplesLinearregressionequationy=0.893x+05.128wasobtainedwithcorrelationcoefficientof0.957821468,AFB11118%,,AFB168217Lgökg8IDC2ELISATLC,r=0.9578,3,2,AFB1AFB1C3,C3BSA,AFB1C3,AFB1,C152C16;,AFG1AFB1;,AFG2AFB1C3,,,AFAF,TLC0721AFB1,(HPLC),TLCAFB1,,,IDC2ELISA,ELISA2,AFB12HSA,,(150000)(2HRP14000),,12.5250pg10%,FanTLIDC2ELISA[6]ELISA,AFB1(10ngömL)Fc,AF,AF,,Fc(),,ELISAChu[6],()10mgömL,,120,,18ELISA;,20mL[10],10ngömL,IDC2ELISATLCr=0.9578,01893,Chu[69]AFB1IDC2ELISATLC,,,AF11,1:,19772ChuFS.HsiaMTS,SunPS,etal.PreparationandcharacterizationofaflatoxionB1212(O2car2boxymethyl)oxime.JAssocOffAnalChem,1977,60(4):7917943DavisMinh2tanB,PrestonJF,etal.Asimplemodifiedcarbodiimidemethodforconjugationofsmall2molecular2weightcompondstoimmunoglobulinGwithminimalproteincrossinglinking.AnalyticalBiochemistry,1981,116:40