梅毒螺旋体TpN15抗原基因的扩增、克隆及表达

目的用基因工程技术表达梅毒螺旋体TpN15重组抗原,为进一步研制梅毒预防性疫苗和诊断试剂盒打下基础.方法采用PCR技术扩增梅毒螺旋体TpN15基因,然后克隆到原核表达载体pET28b中表达TpN15重组抗原蛋白.结果成功的构建了pET28b-TpN15重组表达载体,重组的TpN15蛋白在大肠杆菌BL21中得到稳定表达.结论梅毒螺旋体TpN15基因在大肠杆菌中的成功表达为建立梅毒血清学诊断的新方法奠定了基础.     

探讨梅毒血清学检测方法的应用

梅毒（syphilis）是由梅毒螺旋体（treponemap—allidum,TP）感染引起的性传播疾病,通过人类粘膜或有破损的皮肤直接接触病人感染性病灶而传播,不但引起人类全身性疾病,同时还传播给下一代,因此加强对孕产妇梅毒的早期诊断和及时治疗已成为当前重要问题。     

梅毒螺旋体血清学检测方法的比较

梅毒螺旋体(Treponema pallidum,TP)是性传播疾病梅毒的病原体.梅毒的血清学检测试验根据抗原不同分为非特异性类脂质抗原试验和梅毒螺旋体抗原试验两大类[1].     

三种检测方法对梅毒血清学诊断价值的评价

目的：评价ELISA、RPR、TRUST对梅毒的诊断价值。方法：用ELISA与RPR、TRUST对205例阳性标本和1254例献血员标本检测结果进行对比试验。结果：ELISA明显优于RPR和TRUST法,结果有显著差异性（P〈0．05）。结论：ELISA在输血工作中应用可提高输血安全性。     

梅毒螺旋体抗原基因的克隆、表达及其在临床检验中的应用

目的　克隆、表达并纯化梅毒螺旋体相对分子质量为 47× 10 3 抗原 ,并检测梅毒患者的血清标本。方法　应用基因扩增技术分离此蛋白的全长基因 ,采用基因工程的方法 ,应用融合表达载体pGEX 2T ,重组、克隆得到带有梅毒螺旋体 47× 10 3 特异性抗原基因的重组菌株 ,经大肠杆菌表达系统获得重组融合蛋白。再经亲和层析获得纯品 ,并联合应用ELISA检测梅毒患者的血清标本 30份 ,同时检测非梅毒患者的血清标本 30份。结果　纯化后获得了梅毒螺旋体相对分子质量为 47× 10 3 的特异性抗原。ELISA检测梅毒患者血清结果均阳性。非梅毒患者血清均阴性 ,无非特异性交叉反应。结论　此项方法具有操作简便、准确的特点 ,为临床检测梅毒开辟了新的领域     

梅毒特异性抗体的两种血清学检测方法比较

本文比较梅毒螺旋体明胶颗粒凝集试验法(TPPA)与酶联免疫吸附试验法(TP - EL ISA)两种方法的敏感性和特异性,评价两种方法在梅毒检测中的应用效果及临床意义.结论 发现明胶颗粒凝集试验法(TPPA)检测及酶联免疫吸附试验法(TP - EL ISA)两种检测方法都可以准确地检测梅毒螺旋体抗体,可根据具体情况选择需要的检测方法.     

丙型肝炎病毒抗原检测试剂在临床诊断中的初步应用

本文利用重组丙型肝炎病毒(HCV)多表位复合抗原免疫动物制备抗HCV多克隆抗体,建立HCAgELISA检测方法,对不同的临床样品进行检测,分析不同临床样品HCAg检出率,并与荧光定量PCR结果进行对比。95份HCAb阳性的样品中,51份样品HCAg阳性,阳性率为53.7%;88份重症肝病患者血清中检出HCAg阳性33份,阳性率为37.5%,均明显高于其它样品(P<0.05);73份肝功异常但HCAb阴性血清样品,检出HCAg阳性8份,阳性率为11.0%;HCAg与HCAb之间存在相关性(r=0.5076,P<0.01),HCAg与HCV-RNA符合率为85.7%。HCAg ELISA检测可作为临床HCV诊断的检测指标,为临床早期诊断及治疗预后提供依据。     

重组抗原Em18双抗原夹心酶联免疫吸附试验法检测泡球蚴抗体的建立及初步评价

目的 建立双抗原夹心酶联免疫吸附试验(ELISA)检测泡状棘球蚴抗体的方法.方法 重组抗原Em18(rEm18)作为包被抗原,以辣根过氧化物酶(HRP)标记rEm18抗原为检测抗原,建立检测人血清中泡状棘球蚴抗体的双抗原夹心ELISA方法;应用方阵滴定法对包被抗原、酶标抗原等条件进行优化,并对该系统的灵敏性、特异性、重复性进行初步分析评价.结果 rEm18最佳包被浓度为2.5μg/mL,HRP标rEm18稀释度为1∶800.灵敏度为92％(46/50),特异性为94％(47/50),假阴性率为8％ (4/50),假阳性率为6％ (3/50),批内变异≤9.55％,批间变异≤14.79％,与Em2-ELISA试剂盒检测结果有良好的一致性.结论 本研究成功建立了快速检测泡球蚴特异性抗体的ELISA法,其可以用于人泡球蚴病的早期诊断.     

三种梅毒血清学试验在梅毒诊断中的应用

梅毒是由梅毒螺旋体感染的性传播疾病,具有高度传染性。梅毒诊断主要依据临床表现、镜检病原体和血清学检测。分泌物直接查找病原体,敏感性较低,应用很少。血清学检测在梅毒诊断和治疗中具有重要意义。梅毒的血清学检测方法较多,不同的检测方法有其特殊的临床诊断价值,也各有一定的局限性。为此,笔者用甲苯胺红不加热血清试验(TRUST)、梅毒螺旋体明胶凝集试验(TPPA)和化学发光法(CLIA)对2979份血清进行检测,并对TPPA和CLIA检测结果不一致的标本用免疫印迹法(WB)进行确证试验。     

Evaluation of recomWell Treponema, a novel recombinant antigen-based enzyme-linked immunosorbent assay for the diagnosis of syphilis

Objective   To evaluate the diagnostic performance of an enzyme immunosorbent assay (recomWell Treponema) for the diagnosis of syphilis. The novel recombinant antigens Tpn47, TpN17 and TpN15 were utilized.
Methods   A total of 782 human serum specimens, belonging to four different categories (blood donors, n  = 200; routine laboratory screening for syphilis, n  = 400; syphilis patients, n  = 122; potential cross-reactors, n  = 60), were evaluated to compare the sensitivity and specificity of the recomWell Treponema kit with a standard whole Treponema pallidum cell lysate antigen-based ELISA (Syphilis Screening) and with micro-haemagglutination (MHA-TP).
Results   The overall specificity and sensitivity of the recomWell Treponema IgG was 98.9% and 98.3%, respectively. The specificity and sensitivity of Syphilis Screening ELISA was 98.7% and 98.3%, respectively. The agreement between recomWell Treponema and Syphilis Screening was 100%, 97.8%, 95.9% and 95% among the blood donor specimens, screening samples, syphilis specimens and the potential cross-reactors, respectively. Values of concordance varying from 96.7% to 98.3% were found in the different groups of sera between recomWell Treponema and MHA-TP. In addition, recomWell Treponema demonstrated a good diagnostic performance when used to detect the IgM to T. pallidum . No false-positive sera were identified and, in 17/19 samples from primary infection, an IgM immune response was found.
Conclusions   recomWell Treponema was shown to be a highly specific and sensitive method in all stages of syphilis screening and it can be considered as alternative to other ELISA tests based on native antigen preparations.     

Expression and purification of the recombinant outermembrane protein Tp0453 of Treponema pallidum and its characterization of immuno-competence

ELISA test has been shown to have some advan tages in relation to the tests used for the diagnosisof syphilis because of its easy and quick perfor mance and result readings. With recent develop ment of the gene engineering technology and eluci dation of the whole genome of Nichols strain ofTreponema pallidum, new protein coding openreading frames (ORFs) are available for testing,and the study on the serological tests based on therecombinant protein have been become the focus ofinter…     

HIV-1 gp41重组抗原的表达及其免疫反应性检测

为了在大肠杆菌中表达具有良好免疫反应性的HIV-1gp41重组抗原,本实验运用基因工程技术,经PCR扩增gp41的主要抗原表位序列,BamHⅠ、XhoⅠ双酶切后与E3质粒连接,转化克隆宿主菌DH5α,再提取重组质粒进一步转化表达宿主菌BL21(DE3),经IPTG诱导表达重组蛋白,纯化后标记HRP,通过双抗原夹心酶联免疫方法检测其免疫反应性和特异性。结果表明,获得的HIV-1gp41重组抗原能够与相应抗体特异性结合,与多种无关抗体间无交叉反应,对825份HIV阴性标本检测无错检。检测结果说明该重组抗原具有良好的免疫反应性,在HIV-1抗体诊断试剂中具有潜在的应用价值,为进一步研究gp41抗原奠定了基础。     

Specific and sensitive diagnosis of syphilis using a real-time PCR for Treponema pallidum

A real-time PCR assay with a Taqman probe was developed that targeted the polA gene of Treponema pallidum. The test was validated using an analytical panel (n = 140) and a clinical panel of genital samples (n = 112) from patients attending a sexually transmitted infections clinic. High sensitivities and specificities of 94-100% were achieved using two real-time PCR platforms, the Rotor-Gene and the iCycler. The assay can be completed within 2 h, enabling reporting in <8 h. This fast and robust assay is suitable for implementation in routine laboratories for diagnosing primary syphilis.     

HIV-1 gag基因p17+24重组抗原表达、纯化及抗原性分析

目的获得HIV-1型gag基因p17＋2，4重组抗原，分析其免疫原性和探讨开发诊断试剂的可能性。方法采用PCR技术扩增HIV-1gag基因p17＋p24核酸片段，并克隆入pTreHis2A表达质粒，在大肠杆菌BL21（DE3）株中表达。用Ni—NTA金属螯合层析法纯化目的蛋白，并用免疫印迹法分析纯化蛋白的抗原特异性。结果重组质粒在BL21（DF3）菌株中经IPm诱导表达一个相对分子质量（肘，）约为51×10^3的融合重组蛋白，重组蛋白经HIV-1p17、p24抗原单克隆抗体鉴定，具有抗原特异性。25份HIV阳性血清、180份HIV阴性血清标本初步经l临床验证，具有较高的检出敏感性和特异性。结论成功构建HIV-1型gag基因p17＋24抗原表达载体，表达纯化的p17＋24重组抗原具有较好的抗原性，可用于诊断试剂的开发。     

An improved method of antigen detection on nitrocellulose: in situ staining of alkaline phosphatase conjugated antibody

A biological stain for alkaline phosphatase was applied to detect immune complexes immobilized on nitrocellulose. This method was sensitive and useful for in situ staining of antigens, in dot-immunobinding assays and immunoblots. In addition to its sensitivity, the staining of alkaline phosphatase conjugated antibody was also highly stable, and allowed a permanent record of original immunochemical data. A rapid and simple method is also described using this technique for screening translation products of recombinant clones.     

前列腺特异性抗原EIA试剂盒的研制及应用

目的 建立可定量测定人血清中前列腺特异性抗原（ＰＳＡ）含量的夹心ＥＬＩＳＡ法,研制ＰＳＡ－ＥＩＡ检测试剂盒。方法 从健康男性精液中提取并纯化ＰＳＡ,分别免疫Ｂａｌｂ／ｃ小鼠和山羊制备特异性单克隆抗体和多克隆抗体,并以纯化的ＰＳＡ为标准品,建立可定量测定血清中ＰＳＡ含量的夹心ＥＬＩＳＡ法。在此基础上组装ＰＳＡ－ＥＩＡ试剂盒,对该试剂盒的特异性、灵敏度、精密度、正确性和稳定性等多项指标进行评价。应用该