梅毒螺旋体血凝试剂盒的研制

以超声波处理的梅毒螺旋体为抗原,致敏经醛化、鞣化处理的绵羊红血球,应用血凝试验检测梅毒患者血清中梅毒螺旋体特异性抗体。同时以国际上公认的标准方法——FTA-ABS作为对照试验。共检测1125份血清(其中梅毒血清205份,非梅毒血清920份).两种方法检测结果完全相符,即敏感性皆为100％,血凝试验的特异性为99％。     

磁凝集法梅毒检测试剂检测梅毒螺旋体

目的应用磁凝集法梅毒检测试剂检测梅毒特异性抗体和反应素。方法应用磁凝集法梅毒检测试剂(Tre-ponema pallidum magnetic particle agglutination,TPMPA)检测梅毒阳性血清,分析梅毒阳性血清符合率、梅毒血清抗体滴度、假阳性检出率及交叉反应。结果用TPMPA-A、TPMPA-B试剂检测26份梅毒阳性血清样品,结果均为阳性,与省血液中心和省疾控中心的检测结果的符合率为100%;应用TPMPA-B试剂检测梅毒抗体滴度结果均较用梅毒甲苯胺红不加热血清试验诊断试剂(TRUST)检测结果高2个滴度;用TPMPA-A、TPMPA-B试剂检测正常血清,结果均为阴性,与省血液中心检测结果相符;用TPMPA-A、TPMPA-B试剂检测5份风湿病患者血清和13份慢性肝病患者血清,均未出现交叉反应。结论磁凝集法梅毒检测试剂具有良好的特异性和敏感性,操作简便、省时,可初步用于检测梅毒特异性抗体和反应素。     

梅毒诊断用甲苯胺红不加热血清试验(TRUST)的研究

作者参考文献制备了TRUST抗原。并以目前国内广泛应用的USR抗原作平行对比,检测了3922份血清标本,其中非梅毒患者血清3700份,梅毒患者血清222份,结果显示其特异性、敏感性良好,可以作为梅毒血清学的一种常规试验。     

应用反应素过筛试验(RST)诊断梅毒的研究

参考文献研制了RST抗原，以本所RPR和TRUST抗原为对照，检测254份血清标本，其中梅毒血清54份，非梅毒血清200份。结果表明RST抗原特异性和敏感性良好，反应模式优于TRUST和RPR抗原。     

3种不同方法检测梅毒的结果比较分析

目的 探讨与分析3种不同方法检测梅毒的临床价值。方法 对我院98例梅毒患者与50例健康体检者均分别采用甲苯胺红不加热血清试验(TRUST)、螺旋体明胶颗粒凝集试验(TPPA)、梅毒螺旋体抗体诊断试剂盒(胶体金法)检测血清梅毒,对3组检测方法的结果进行比较分析。结果 TRUST的试验检验符合率为86.7%,TPPA的试验检验符合率为100%,胶体金的试验检验符合率为99.0%。TPPA与TRUST相比(χ2=13.92,P<0.01)有统计学差异。胶体金与TRUST相比(χ2=11.08,P<0.01)有统计学差异。结论 胶体金法的检出率明显优于TRUST,但与TPPA相比则无明显差异,故胶体金法在对梅毒的检测中比TRUST的准确率高,比TPPA更易操作,且成本较低。     

ELISA在梅毒检测中的价值

近年来梅毒发病率正呈逐年增长之势 ,为了解两种常用梅毒血清诊断方法的差异 ,本文对ＴＲＵＳＴ和梅毒 ＥＬＩＳＡ两种方法进行了比较。取血浆样本 2 0 70份 ,经ＴＲＵＳＴ检测阳性 90份 ,阴性1980份 ,再经上海科华生物工程股份有限公司的梅毒螺旋体抗体酶联免疫法诊断试剂盒复检 ,结果不一致的样本再用日本富士公司的ＴＰＰＡ试剂盒作进一步检测。结果 90份ＴＲＵＳＴ阳性标本中有 3份梅毒 ＥＬＩＳＡ为阴性 ,1980份ＴＲＵＳＴ阴性标本中 ,12份为阳性 ,共有 15份不一致标本。用ＴＰＰＡ试剂检测这 15份标本 ,结果 3份梅毒 ＥＬＩＳＡ阴性标…     

梅毒血清试验检测在诊断梅毒中的作用和监控效果

目的探讨梅毒血清学试验检测方法在诊断梅毒中的作用和监控效果。方法回顾性分析我院收治的健康体检人员血清44例、各期梅毒血清98例、非梅毒血清37例进行TRUST、TP-ELISA、TPPA检测。结果 TRUST、TP-ELISA检测结果和TPPA相比均有统计学意义(P<0.05),检测中以TRUST检测的阳性率和敏感性最低,TP-ELISA检测明显高于TRUST。结论 TRUST、TP-ELISA、TPPA都是检测确诊梅毒的重要方法。选择合适的检查方法,有效预防和控制梅毒,有利于血液质量的保证,提高患者生命。     

梅毒螺旋体特异性抗原TP17、TP0453的表达及以其作为诊断抗原的间接ELISA方法的建立

目的表达、纯化梅毒螺旋体特异性外膜蛋白TP17和TP0453,并以其作为包被抗原,建立新型梅毒酶联免疫诊断方法。方法 PCR扩增TP17和TP0453基因,分别克隆入pET-22b(+)和pET-28a(+)载体,构建重组表达质粒pET-22b(+)-TP17和pET-28a(+)-TP0453,转化E.coliBL21(DE3),IPTG诱导表达,表达产物纯化后进行Western blot鉴定。以纯化的重组TP17和TP0453蛋白作为诊断抗原,建立间接ELISA方法,并对该方法进行验证。结果 PCR分别扩增出500和800bp的TP17和TP0453基因片段,测序结果与GenBank中登录的CDS序列完全一致。重组表达质粒pET-22b(+)-TP17和pET-28a(+)-TP0453经双酶切鉴定正确;重组TP17和TP0453蛋白的相对分子质量分别约为18000和29000。表达量分别约占菌体总蛋白的18%和24%,纯化的重组TP17和TP0453蛋白的纯度分别>95%和>90%。两种重组蛋白均能与梅毒标准血清发生特异性反应。检测70份梅毒参考血清和定值质控血清的批内变异系数(CV)≤4.35%,批间CV≤6.69%,表明精密性良好;对国家标准血清盘的检测灵敏度为94.4%,特异性为97.1%,符合率为95.7%,最低检出限为0.25NCU/ml。结论表达和纯化的重组TP17和TP0453蛋白具有良好的抗原性和特异性,可用于梅毒血清学诊断。     

胶体金法试剂与EIA试剂检测梅毒螺旋体抗体的临床比较分析

目的探讨胶体金法梅毒螺旋体(TP)抗体试剂的灵敏度与特异性。方法用胶体金法与EIA法对188份梅毒患者血清样本进行检测。结果TP抗体S/CO值在>10.0时,胶体金法与EIA法的阳性符合率只有85.3%。结论胶体金法检测TP抗体S/CO值高值样本时存在着一定的漏检率。     

流感嗜血杆菌感染反向血凝诊断试剂盒的研制

应用自制的流感嗜血杆菌（Hi）血清提取的免疫球蛋白致敏醛化血球制备的反向血凝诊断试剂盒，与美国Difco血清制备的试剂盒相比，其灵敏度无差异，均可达2～4ng／25μl，而特异性较好。应用该试剂盒检测了152份正常人血清，29份肺炎病人血清，311份尿标本（其中55份为正常儿童尿，256份为肺炎病人尿），60份肺炎病人痰标本。正常人标本检测结果均为（一），病人血清、尿、痰标本阳性率分别为13％、7％和10％。     

梅毒螺旋体特异性抗原TpN47基因片段的克隆与表达

目的在大肠埃希菌中表达梅毒螺旋体(Tp)TpN47(68~410 aa)重组蛋白。方法采用PCR法从Tp全基因组中扩增TpN47(202~1230 bp)片段,T-A克隆后构建原核表达质粒pET-22 b(+)-TpN47,经酶切鉴定后转化EcoliBL21(DE3),IPTG诱导表达,表达产物经Ni2+亲和层析柱纯化,Western blot鉴定其免疫反应性。结果PCR法扩增出约1 000 bp的目的片段,原核表达质粒pET-22 b(+)-TpN47经酶切鉴定正确,表达的蛋白约占菌体总蛋白的43%,相对分子质量约为39 000,以包涵体形式存在。经纯化后蛋白纯度达95%以上,Western blot证实该蛋白能与梅毒患者阳性血清发生特异性反应。结论所表达的TpN47重组蛋白具有良好的免疫反应性,为开发临床检测效果更好的梅毒诊断试剂盒奠定了实验基础。     

妊娠晚期胎死宫内的临床研究

目的 探讨妊娠晚期胎死宫内的原因及预防方法。方法 对我院2009年5月至2011年5月收治的妊娠28周后胎死宫内的患者48例的临床资料进行回顾性分析。结果 妊娠合并症:妊娠高血压综合征22例,梅毒8例,贫血2例,胎儿横位2例。脐带因素:脱垂20例,扭转8例,缠绕4例。胎盘因素:早剥14例,过期6例。其他:不明原因6例。多数患者有以上病因数种并存。阴道分娩38例,因胎盘早剥而行剖宫取胎10例。结论 应加强对妊娠晚期孕妇的引导,对出现的异常情况及时处理以降低死胎比率。     

A Novel Quantum Dots–Based Point of Care Test for Syphilis

One-step lateral flow test is recommended as the first line screening of syphilis for primary healthcare settings in developing countries. However, it generally shows low sensitivity. We describe here the development of a novel fluorescent POC (Point Of Care) test method to be used for screening for syphilis. The method was designed to combine the rapidness of lateral flow test and sensitiveness of fluorescent method. 50 syphilis-positive specimens and 50 healthy specimens conformed by Treponema pallidum particle agglutination (TPPA) were tested with Quantum Dot-labeled and colloidal gold-labeled lateral flow test strips, respectively. The results showed that both sensitivity and specificity of the quantum dots–based method reached up to 100% (95% confidence interval [CI], 91–100%), while those of the colloidal gold-based method were 82% (95% CI, 68–91%) and 100% (95% CI, 91–100%), respectively. In addition, the naked-eye detection limit of quantum dot–based method could achieve 2 ng/ml of anti-TP47 polyclonal antibodies purified by affinity chromatography with TP47 antigen, which was tenfold higher than that of colloidal gold–based method. In conclusion, the quantum dots were found to be suitable for labels of lateral flow test strip. Its ease of use, sensitiveness and low cost make it well-suited for population-based on-the-site syphilis screening.     

Development of a Method of Measuring β-D-Glucan and Its Use in Preemptive Therapy for Invasive Fungal Infections

Invasive fungal infections (IFIs) are serious infections that develop in conjunction with neutropenia after chemotherapy for acute leukemia or with hematopoietic stem cell transplantation. Conventionally, empirical antifungal therapy was recommended to treat IFIs for patient safety despite a lack of evidence of fungal infections. However, many studies have indicated that antifungals were not necessary for over half of patients, and several detriments of empirical therapy were noted, e.g., antifungals caused adverse reactions, an increase in drug-resistant fungi was a possibility, and medical costs soared. β-D-glucan (BDG) is a component of clinically important fungi such as Aspergillus and Candida. The G-test was developed in Japan as a way to measure BDG in serum using a coagulation factor from the blood of the horseshoe crab. Pre-emptive antifungal therapy based upon serodiagnosis with a BDG or galactomannan assay and CT imaging has been introduced. With pre-emptive antifungal therapy, the prognosis is equivalent to that with empirical therapy, and the dose of the antifungal has been successfully reduced. Measurement of BDG has been adopted widely as a method of diagnosing IFIs and is listed in the key guidelines for fungal infections and febrile neutropenia.     

Phage Display Screening of Bovine Antibodies to Foot-and-Mouth Disease Virus and Their Application in a Competitive ELISA for Serodiagnosis

For serodiagnosis of foot-and-mouth disease virus (FMDV), monoclonal antibody (MAb)-based competitive ELISA (cELISA) is commonly used since it allows simple and reproducible detection of antibody response to FMDV. However, the use of mouse-origin MAb as a detection reagent is questionable, as antibody responses to FMDV in mice may differ in epitope structure and preference from those in natural hosts such as cattle and pigs. To take advantage of natural host-derived antibodies, a phage-displayed scFv library was constructed from FMDV-immune cattle and subjected to two separate pannings against inactivated FMDV type O and A. Subsequent ELISA screening revealed high-affinity scFv antibodies specific to a serotype (O or A) as well as those with pan-serotype specificity. When BvO17, an scFv antibody specific to FMDV type O, was tested as a detection reagent in cELISA, it successfully detected FMDV type O antibodies for both serum samples from vaccinated cattle and virus-challenged pigs with even higher sensitivity than a mouse MAb-based commercial FMDV type O antibody detection kit. These results demonstrate the feasibility of using natural host-derived antibodies such as bovine scFv instead of mouse MAb in cELISA for serological detection of antibody response to FMDV in the susceptible animals.