甲胎蛋白化学发光免疫定量分析方法的建立.doc

PAGE PAGE 1 甲胎蛋白化学发光免疫定量分析方法的建立 【摘要】目的：建立检测甲胎蛋白化学发光免疫定量分析方法.方法：采用双抗体异位点一步夹心法，以甲胎蛋白单克隆抗体作为固相包被，采用改良过碘酸钠法进行甲胎蛋白多克隆抗体与碱性磷酸酶偶联制备酶标抗体；以金刚烷胺衍生物CSPD作为底物，优化CSPD与SapphireII发光体系；用国家标准品校定定量标准品，建立了人血清AFP的化学发光免疫定量分析法.用该法检测800份正常人血清，确定正常值参考范围，将临床血清标本100份与bayerAcs:180j全自动发光体系统进行比较.结果：本方法的最低检出量为2.0ng/mL，线性范围2.0~600.0ng/mL,批内和批间的变异率分别为6.7%和7.7%，回收率在98.2%~102.5%之间.与CEA(3μg/mL)的交叉≤0.15%；与铁蛋白(10μg/mL)≤0.04%；与人血清白蛋白（200g/L）≤2.5×10-8，与人血清丙球蛋白（100g/L）≤2.0×10-8，与bayerAcs:180j比较相关系数r=0.994（Plt;0.001）.结论：建立的甲胎蛋白化学发光免疫分析法可常规用于临床检测. 【关键词】甲胎蛋白类；化学发光测定法；定量分析 　　Establishmentofchemiluminescentimmunoassayfordeterminationofalpha瞗etoprotein 　　FENGYan睲ing,MAJun睩en,WUBiao,GUOLan瞃ing,WUYong睯un,LIZhi睺ao,WUYi睲ing 　　1DepartmentofLabourHealthandOccupationalPublicHealthInstitution,ZhengzhouUniversity,Zhengzhou450052,China,2Sino睞mericanBiologyCompany,Luoyang,471003,China 　　【Abstract】AIM:Toestablishamethodofchemiluminescentimmunoassayfordeterminationofalpha瞗etoprotein(AFP)inhumanserum.METHODS:Thetwo瞫iteenzymeimmunoassayisbasedonthedirectsandwichtechnique.AmonoclonalantibodywasbondedtothemicroplateforcoatingandtheconjugationofalkalinephosphatasetoanotherpolyclonalantibodywasperformedbyNaIO4method.ChemiluminescentimmunoassaywasestablishedbyoptimizingCSPDandSapphireIIluminescentsystem.Sensitivity,linearscope,precision,interferenceandrecoveryforthemethodwereevaluated.Thereferencerangewasdefinedbytesting800serumsamplesfromhealthysubjects.Thecomparisonfor100serumsamplesfrompatientswasassayedbythebayerAcs:180jAutomatedImmunoassaysystem.RESULTS:Thedetectableminimumwas2.0ng/mL.Thelinearscopewasfrom2.0to600.0ng/mL.Averageinterandintra瞐ssaywere7.7%and6.7%,respectively.Therecoveriesrangedfrom98.2%to102.5%.Thecross瞨eactingrateforCEA(3μg/mL),Ferritin(10μg/ml),HAS(200g/L),IgG(100g/L)were≤0.15%,0.04%,2.5×10-8and2.0×10-8,respectively.ComparedwithbayerAcs:180jAutomatedimmunoassaysystem,therelativecoefficientwas0.994（Plt;0.001）.CONCLUSION:Asuccessfulestablishmentofchemiluminescentimmunoassayprovidesawayforclinicaldetermin