摘要
目的建立检测抗－ＨＣＶ抗体的双抗原夹心酶联免疫吸附测定（ＥＬＩＳＡ）方法。方法用基因工程手段，在大肠杆菌中表达ＨＣＶ抗原表位与大肠杆菌噬菌体ＭＳ２ＤＮＡ聚合酶（ＭＳ２－Ｐｏｌ）的融合蛋白，纯化后作为包被抗原，可溶性表达的ＨＣＶ抗原表位与霍乱毒素Ｂ亚基（ＣＴＢ）的融合蛋白经辣根过氧化物酶标记后作为酶结合物，建立双抗原夹心ＥＬＩＳＡ。结果ＭＳ２－Ｐｏｌ融合蛋白在大肠杆菌中得到高效表达并进行了纯化，所建立的双抗原夹心ＥＬＩＳＡ系统用于检测１４份ＨＣＶ阳性血清样品，抗－ＨＣＶ抗体检出率１００％；检测２１份ＨＣＶ阴性血清，没有假阳性结果。ＣＴＢ抗体不干扰反应。结论双抗原夹心法与采用二抗的ＥＬＩＳＡ系统具有相近的灵敏度和更好的特异性。可以避免由于二抗干扰带来的非特异性。
ObjectiveToestablishanELISAsystemforthedetectionofanti-HCVantibodiesbasedonantigen-sandwichedprinciple.MethodsBothrecombinantfusionantigenofcholeratoxinBsubunit(CTB)-HCVepitopesandcoliphageMS2DNApolymerase-HCVepitopeswereexpressedinE.coliasasolubleformoraninclusionbodyform.PurifiedMS2-HCVfusionproteinwasusedassolid-phaseantigenandthesolubleCTB-HCVfusionantigenusedasconjugatesafterbeingconjugat-edwithhorseradishperoxidase.ResultsFusionproteinofcoliphageMS2DNApolymeraseandHCVepitopeswereexpressedinE.coli.ThefusionproteinsretainedthereactivityofHCVepitopes.AntigensandwichedELISAshowed100%sensitivityindetecting14HCVpositivespecimensand100%specificityin21HCVnegativespecimens.ConclusionsThemethodestablishedretainedthesamesensitivityandbetterspecificityascomparedtoroutinemethodinwhichthesecondantibodywasusedasalabel.
