石杉碱甲人工抗原的合成,鉴定及免疫原性分析

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1、石杉碱甲人工抗原的合成鉴定及免疫原性分析宋建武1，晁　志2*，董为人1南方医科大学1基础医学院组织学与胚胎学教研室，2中医药学院，广东广州510515摘要：目的　制备乙酰胆碱脂酶抑制活性成分石杉碱甲（HuperzineA，HupA）的人工抗原及抗血清，为制备HupA的单克隆抗体、建立快速检测HupA的酶联免疫吸附测定（ELISA）法提供技术基础。方法　将HupA与戊二醛、载体蛋白牛血清白蛋白（BSA）反应偶联，制得半抗原－载体蛋白复合物后，用基质辅助激光解吸飞行时间质谱测定其中结合的半抗原数目；以此抗原免疫BALB/c小鼠，制备抗血清，并通过ELISA法检测其抗体效价和特异性。结果　合

2、成的人工抗原HupA-GA-BSA中HupA与BSA的结合比约为8:1；免疫小鼠得到特异针对HupA的抗血清，HupA抗体的效价为1:6250。结论　成功地合成了HupA的人工抗原，且该抗原有较好的免疫原性。关键词：石杉碱甲；半抗原-载体蛋白复合物；免疫原性；基质辅助激光解吸飞行时间质谱；酶联免疫吸附测定SynthesisofhuperzineA-bovineserumalbuminconjugateanditsimmunogenicityanalysisinmiceSONGJian-wu1,CHAOZhi2,DONGWei-ren1DepartmentofHistologyandEm

3、bryology,CollegeofBasicMedicine1,SchoolofTraditionalChineseMedicine2,SouthernMedicalUniversity,Guangzhou510515,ChinaAbstract:ObjectiveTopreparehuperzineA-carrierproteinconjugateanddetermineitsimmunogenityforlaterproductionofmonoclonalantibodyagainstHupAandestablishinganenzyme-linkedimmunosorbent

4、assay(ELISA)-baseddetectionmethodofHupA.MethodsHupAwasconjugatedwithbovineserumalbumin(BSA)usingglutaraldehyde(GA)methodtoobtaintheHupA-GA-BSAconjugate,andthehaptennumberintheconjugatewasdeterminedbymatrix-assistedlaserabsorptionionizationtime-of-flightmassspectrometry(MALDI-TOF-MS).BALB/cmicewe

5、reimmunizedwithHupA-GA-BSAtopreparetheantiserumagainstHupA.TheserumtiterandspecificityoftheHupAantibodiesweredetectedbyindirectELISAandcompetitiveELISA,respectively.ResultsTheconjugationratioofHupAandthecarrierproteinBSAwas8:1.TheantiserumagainstHupAwithatiterof1:6250wasobtainedafterimmunizingth

6、emicewiththeconjugate,andtheantiserumreactedspecificallytoHupA.ConclusionThesynthesizedHupA-GA-BSAconjugatepossessesgoodimmunogenityinmice,suggestingthefeasibilityofpreparingthemonoclonalantibodyagainstHupAusingthisconjugate.Keywords:HuperzineA;hapten-carrierproteinconjugate;immunogenity;matrix-

7、assistedlaserabsorptionionizationtime-of-flightmassspectrometry;enzyme-linkedimmunosorbentassay作者简介：宋建武，男，讲师，硕士研究生。*通讯作者：晁志，男，博士，副教授，电话：020-61648256.E-mail:chaozhi1971@yahoo.com.cn石杉碱甲（HuperzineA，HupA）是从中国石杉科石杉属蛇足石杉（Huperzia