摘要
目的 寻找高灵敏度、高特异性、检测结果定量、简便易行的梅毒螺旋体抗体酶联免疫检测方法。 方法 采用重组技术 ,在现有已公布的梅毒螺旋体基因组中筛选出公认抗原性最强的三个基因片段 Tpp1 5、Tpp1 7、Tpp47进行克隆。为了获得水溶性高、特异性强、可标记性好的重组抗原蛋白 ,有针对性地设计引物以便对表达的梅毒螺旋体膜蛋白进行修饰和改造 ,同时在收获高效表达的目的蛋白后采取了一系列针对性切割和高特异性纯化。以纯化的重组梅毒螺旋体膜蛋白 (r-KTp1 5 ,r-KTp1 7及 r-KTp47)为抗原 ,建立了诊断梅毒螺旋体抗体的双抗原夹心酶联检测方法 (DAGS ELISA)。 结果 在大肠杆菌中获得了重组梅毒螺旋体膜蛋白的高效表达 ,经修饰纯化之后的目标蛋白水溶性提高 ,特异性增强 ,可标记性优于常规不修饰的重组蛋白。应用该三种蛋白为抗原建立的双抗原夹心法血清学诊断试剂盒共检测各期梅毒阳性患者 2 1 0例 ,灵敏度达 98.6% (2 0 7/2 1 0 ) ,检测正常献血者 1 3 2 7例 ,特异性达 1 0 0 .0 % (1 3 2 7/1 3 2 7) ,优于常规梅毒血清学初筛诊断方法 RPR及 TRUST法 ,与 TPHA法的符合率为 1 0 0 .0 %。 结论 以双抗原夹心法酶联免疫诊断试剂盒在梅毒血清学初筛实验室进行梅毒螺旋体抗体的筛查 ,在灵敏度、特异?
Objective To study a one step double antigen sandwich enzyme linked immunosorbent assay (ELISA) procedure on detecting treponema pallidum antibody. Methods The serum of syphilitics and appropriately diluted peroxidase (HRP) conjugated with recombinant treponema pallidum (r TP) antigen are filled together into the microplate which coated well with same modified recombinant treponema pallidum antigen.Then the treponema pallidum antibodies can be bounded by HRP TP and the same recombinant treponema pallidum antigen.We optimized the pH and the recombinant concentrations to coat the microplate and the dilution of the peroxidase (HRP) conjugated with treponema pallidum antigen.The time of the one step reaction and HRP substrate reaction are optimized also. Results\ Serum specimens from 210 syphilitics patients and 1 327 non infected subjects were investigated.The sensitivity and specificity of treponema pallidum one step double antigen sandwich (DAGS) enzyme linked immunosorbent assay (ELISA) were comparable to those of treponema pallidum haemagglutination assay (TPHA) and the flouresent treponemal antibody absorption (FTA) test. Conclusion The ELISA test support the potential consideration of the assay as a confirmatory test for the serodiagnosis of syphilitics.
出处
《临床输血与检验》
CAS
2002年第3期10-14,共5页
Journal of Clinical Transfusion and Laboratory Medicine
