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:1007-8738(2006)01-0121-041,1,2,3,13(1,510515;2,510663;3,100050):2005-01-20;:2005-06-20:(No.2003U130021):(1976-),,,,.3Correspondingauthor,Tel:(020)61648550Email:mingli@fimmu.comTime2resolvedfluoroimmunoassayofcarcino2embryonicantigenandprepa2rationofitsdiagnosticreagentHANGJian2feng1,WUYing2song1,YUWei2hong2,HUANGYing3,LIMing131SchoolofBiotechnology,SouthernMedicalUniversity,Guang2zhou510515;2GuangzhouDaruiantibodyCo.,Ltd.,Guangzhou510663;3NationalInstitutefortheControlofPharmaceuticalandBiologicalProduces,Beijing100050,China[Abstract]AIM:Toestablishatwo2sitetime2resolvedfluoroimmunoassay(TRFIA)fordetectionofcarcino2embry2onicantigen(CEA)basedonthedirectsandwichtechnique(Onemonoclonalantibody(mAb)isimmobilizedonthesur2faceofmicrotiterplatestripwells,anothermAbislabelledwithEu3+)andprepareitsdiagnosticreagent.METHODS:ThesandwichTRFIAfordetectionofserumCEAlevelusinganti2CEAmAbs.RESULTS:ThemeasurerangeofCEA2TRFIAwas(1-560)g/L.Theanalyticalsensitivitywas0.28g/L.Theintra2andinter2assaycoefficientsofvaria2tion(CV)were7.2%-8.6%and8.9%-13.2%,respec2tively.Therewasnocross2reactiontoAFP,CA1225,CA1929andalbumin.CrossreactivitytoCA1523was1.62g/L.ThecorrelationcoefficientofbloodsamplesdetectedbyCEA2TRFIAandcommerciallyavailableCEACLIA(chemilu2minescentimmunoassay)kit(Roche)was0.946.CON2CLUSION:CEA2TRFIAisanewimmunoassaywhichhasseveraladvantagesincludinghighsensitivity,specificityandawideworkingrange.Itissuitableforroutineclinicaluse.[Keywords]carcino2embryonicantigen(CEA);time2re2solvedfluoroimmunossay(TRFIA)[]:(TRFIA),(CEA):(1CEAmAbM86160M,1CEAmAbM86110M)CEA2TRFIA,2:CEA2TRFIA(1560)g/L,0.28g/L,(CV)7.2%8.6%8.9%13.2%AFPCA1225CA1929,CA15231.62g/L1000,(r)0.946:CEA2TRFIA,,CEA[];[]R392.33[]B,(CEA),[1]CEA1965GoldFreedman,(Mr)220000,,CEA,,CEA,,[2](TRFIA)[3,4],,(),,,TR2FIA3CEA2TRFIA,CEA11.1Eu3+CEAmAbM86160MM86110MCEAAFPCA1225CA192121ISSN1007-8738(ChinJCellMolImmunol)2006,22(1)9CA1523,Biodesign96(812)LabsystemSephadexG250PharmaciaEu3+Eu3+,Per2KinElmerCEA,WallacDELFIATMCEA(RocheCEAkit)2PerkinElmer1000(CEA556,444)425,301MilliporeAuto2Delfia1235,2PerkinElmer1.21.2.150mmol/LpH9.6,mAbM86160M3mg/L,96,200L,4,(20g/LBSA),250L,372h,,-201.2.2Eu3+mAb0.5mgmAbM86110M,8000r/min56min(50mmol/LNa2CO3,pH9.5)6,200LmAb0.5mgEu3+,41.2.3Eu3+mAbM86110MmAbSephadexG250(1cm30cm),(9g/LNaCl50mmol/LTris2HCl)(1mL/),,,Eu3+Eu3+Eu3+mAb1.2.42g/LBSA1g/LNaN350mmol/LpH7.8Tris2HCl,CEA1510100560g/L,1mL,-201.2.5,25LCEA,200L(5g/LBSA9g/LNaCl1g/LNaN350mmol/LpH7.8Tris2HCl),3h(9g/LNaCl0.2g/LNaN30.3g/LTween8050mmol/LpH7.8Tris2HCl)4,175Eu3+mAb200L,1h6,200L5minAuto2Delfia12351.2.6CEA2TRFIA(1):,,2tCEA,(2):(3):(A1)20,A12,,(4):3(2565160g/L),,8,CV(5):AFPCA1225CA1523CA1929,(6):CEA2TRFIA425,CEASPSS11.5(7):CEA2TRFIA,CEA30,,,RocheCEAkit1000(556,444),22.1mAbM86110MmAbM86110M9.58%86%2.2,(log2log),2(1A:-;1B:-;Cps),0.4620.455(tP0.05)CEA,1(0.91.1)2(r)=0.9981CEA2TRFIA-Fig1Accuracyofdose2responsecurveforCEA2TRFIAA:Dose2responsecurvefornationalstandard;B:Dose2responsecurveforreferencestandard.2.31560g/LA12,0.28g/L,CEA221ISSN1007-8738(ChinJCellMolImmunol)2006,22(1)(CEA0.2g/L)2.4CEACEA2TRFIA,CV10%,CV15%(2)1CEA2TRFIA-Tab1Accuracyofdose2responsecurveforCEA2TRFIAreagent(n=8)ItemNS(g/L)0(A)1(B)5(C)10(D)100(E)560(F)RS(g/L)0.06(A1)00.11.06(B1)0.941.085.25(C1)4.95.39.62(D1)9.710.7104(E1)92106545(F1)519583Ratio(RS/NS)-1.060.941.081.050.981.060.960.971.070.960.921.061.040.931.04NS=nationalstandard;RS=referencestandard.A-F:Concentrationofnationalstandard;A1-F1:Concentrationofreferencestandard.2CEATab2Accuracydetectedbyself2madeCEAreagent(n=8)GroupIntra2assayLotNo.040225CV(%)LotNo.040405CV(%)LotNo.040620CV(%)Inter2assay3lotsCV(%)QCL8.980.687.69.120.717.89.480.768.09.291.2313.2QCM26.82.178.127.92.408.628.22.177.728.62.548.9QCH83.86.037.285.26.397.584.96.968.286.58.399.7QC:Qualitycontrol;L:Lowconcn;M:Mediumconcn;H:Highconcn.2.5CEA1106U/LAFP6105U/LCA12255105U/LCA15234105U/LCA19291106g/L,CEA2TRFIA,CEA001.6200g/L2.6ROCCEA2TRFIA425(229,391;196,890)CEA,0g/L,10.3g/L,(x)2.76g/L,(SD)1.88g/L32,(95.53%)CEA6.0g/L,2SD6.52g/L;96.71%CEA6.5g/L,,CEA06.5g/L,,1000CEA2TRFIA,SPSS11.5,ROC,ROC0.977(95%0.9690.985),CEA3CEATab3StatisticalanalysisofdetectingresultsofserumCEAlevelinhealthyindividuals(n=425)IndexCEAlevel(g/L)0-6.06.01-7.07.01-8.08.01-10.010.01-12.012.0Samplenumber40673810Ratio(%)95.531.650.701.880.240Percentage(%)95.5397.1897.8899.76100-2CEAROCFig2NormalrangeofserumCEAlevelinhealthyindividualsandROCcurveA:NormalcurveforCEA2TRFIA;B:ROCcurveforCEA2TRFIA.2.7CEA(30)CEA2TRFIA,3029;3029,96.7%CEA2TRFIACEA,Wallac2CEARocheCEAkitCEA2TRFIA,1000(CEA556,444),r0.946(43)321ISSN1007-8738(ChinJCellMolImmunol)2006,22(1)4CEAWallac2CEATab4Comparisondetectingresultsbetweenself2madeCEArea2gentandWallac2CEAkitIndexSelf2madeCEAreagentWallac2CEAkitSensitivity(g/L)0.280.2Linearrange(g/L)15601500Precision(%)CV8.6CV5Stability1yearat41yearat43CEARoche2CEACEA()Fig3Correlation(regressionanalysis)ofserumCEAdetectedbyself2madeCEAreagentandRocheCEAkit(r=0.946)3TRFIA,[5-7],,,,(),,(),TRFIA(,103106Stokes290nm;Stokes28nm),,,CEA,[8],CEA20g/L,;,15%53%