免疫-PCR法检测梅毒螺旋体特异性抗体

以梅毒螺旋体重组蛋白为抗原,应用免疫-PCR方法检测梅毒螺旋体抗体,并同常规ELISA法进行比较,探讨免疫-PCR方法检测梅毒螺旋体特异性抗体的可行性。结果免疫-PCR法敏感性是常规ELISA法的104倍,阳性检出率高于ELISA法;对照血清标本梅毒螺旋体抗体检测为阴性。表明免疫-PCR方法具有较高敏感性和特异性,有一定的临床推广价值,对梅毒患者的早期诊断及时治疗等具有重要意义。     

胶体金法与TPPA法检测梅毒特异性抗体的对比研究

目的 :探讨梅毒螺旋体特异性抗体胶体金法 (TPAb)在临床工作中的应用。方法 :以梅毒累旋体明胶凝集试验 (TPPA)为“金标准” ,同时用TPAb法检测梅毒高危人群的血清。结果 :两种方法经 χ2 检验P >0 0 5 ,差异没有显著性 ,Kappa值为 0 86 ,TPAb法其敏感性 88 9% ,特异性 97 8% ,准确性 93%。结论 :TPAb法可推荐用于血清学梅毒特异性抗体检查 ,有助于临床梅毒的快速诊断     

Immuno-PCR法诊断早期梅毒的方法学建立及其应用

目的建立Immuno-PCR法诊断早期梅毒的方法学,评价其灵敏度、特异性、重复性及其临床应用。方法利用基因重组TpN47抗原免疫新西兰兔,制备抗体并用Weston blotting检测;利用抗TpN47抗体作为捕获抗体与血清中TpN47抗原结合,通过链霉亲和素、生物素化抗体、生物素化DNA和PCR扩增等建立Immuno-PCR法检测梅毒螺旋体抗原TpN47体系;评价该方法的灵敏度、特异性和重复性;收集200例临床标本通过Immuno-PCR法、ELISA、TPPA和TURST法进行临床应用比较。结果 Weston blotting结果显示TpN47抗体阳性;Immuno-PCR比ELISA法敏感性强103倍,比TPPA、TURST强105倍;特异性高,重复性好。临床标本中Immuno-PCR法敏感性和特异性分别为86.00%（P〈0.05）和100.00%,ELISA法为71.00%和98.00%,TPPA法为65.00%和100.00%,TRUST法为68.00%和95.00%。结论 Immuno-PCR法检测梅毒螺旋体TpN47抗原敏感性高,特异性强,重复性好,可作为梅毒螺旋体感染的早期诊断方法 。     

电化学发光法检测梅毒螺旋体特异性抗体的应用研究

目的对罗氏电化学发光免疫法(ECLIA)检测梅毒螺旋体特异性抗体的临床价值进行评估。方法收集梅毒疑似病例血清标本132份,分别用ECLIA、梅毒螺旋体明胶凝集试验(TPPA)和免疫印迹法(WB)进行检测,以WB为金标准,计算并比较ECLIA和TPPA的灵敏度和特异性,进而比较化学发光免疫法检测低S/CO值和高S/CO值的灵敏度和特异性差异。结果针对132份血清标本,ECLIA敏感性为100.00%,特异性为83.33%,阳性预测值为96.43%,阴性预测值为100.00%,总符合率为96.97%。TPPA敏感性为93.52%,特异性为87.50%,阳性预测值为97.12%,阴性预测值为75.00%,总符合率为92.42%。ECLIA检测1≤S/CO3组与S/CO≥3组的敏感性均为100.00%,特异性分别为86.96%和95.24%,结论 ECLIA检测具有较高的敏感性,适合临床大样本筛查,对S/CO值低的标本应结合TPPA、WB及临床资料确诊。     

化学发光微粒子免疫法和梅毒螺旋体颗粒凝集试验检测梅毒抗体的比较

目的评价化学发光微粒子免疫法（chemiluminescence microparticle immunoassay, CMIA）检测临床血清标本梅毒螺旋体抗体的敏感性和特异性。方法用梅毒螺旋体颗粒凝集试验（TVeponema Pallidum particie agglutination test,TPPA）法作为对照标准,采用CMIA法检测2012年11月到12月1200例住院患者的血清标本,并用卡方检验评价两种检测方法对同一个样本的化验结果的一致性。结果1200例血清标本中用CMIA法检出阳性率为11.3%,TPPA法检出阳性率为10. 9% ,以TPPA为标准,CMIA法敏感性为96. 9%,特异性为99. 2%,其中CMIA法检测血清S/CO值〉 4. 00的110例,用TPPA确认107例阳性,阳性预测值（PPV）为97.3%;CMIA法S/C0值在1.0-9.0,TPPA可出现阴性结果。结论CMIA法可替代TPPA法进行梅毒螺旋体抗体检测,对于CMIA法检测S/C0值1. 0-4.0的需进一步复检。     

西门子ADVIA Centaur XP化学发光免疫分析仪检测梅毒螺旋体特异性抗体的测量阈值建立及结果比较

本研究旨在探讨西门子ADVIA Centaur XP梅毒血清学试验化学发光免疫分析法 (CLIA) 检测梅毒螺旋体特异性抗体的测量阈值,并与化学发光微粒子免疫分析法 (CMIA)、梅毒螺旋体免疫印迹法 (TP-WB) 及梅毒螺旋体抗体颗粒凝集试验 (TPPA) 法检测结果进行比较,为CLIA方法的规范应用提供参考。收集河北燕达医院2018年7月至2019年7月雅培ARCHITECT i2000 CMIA法检测梅毒螺旋体特异性抗体筛查样本30 887例,选取CMIA法筛查梅毒特异性抗体中样品吸光度与临界值的比值 (S/CO) 为1–9的153例患者为研究对象,采用梅毒血清学检测逆序流程,并分别以TP-WB及TPPA法为确认方法,运用MedCalc对结果进行受试者工作特征曲线 (ROC曲线) 分析,获得截断值,采用卡方检验进行计数资料的差异显著性检验。采用不同方法对同一批血清样本梅毒螺旋体特异性抗体的检测结果存在差异,CLIA法与TPPA法差异不显著,与TP-WB法及CMIA法差异显著;分别以TPPA检测结果和TP-WB检测结果为金标准,ROC曲线分析发现,CLIA法的最佳诊断截断值分别为4.01和16.06,曲线下面积分别为0.961和0.838。采用不同梅毒血清学试验方法作为金标准时,CLIA法建议截断值差异较大,因此,CLIA测定梅毒抗体S/CO为1.00–16.06时,实验室血清学检测应推荐首选TP-WB方法学进行复核确证,避免临床误诊发生。     

基因重组抗原检测梅毒螺旋体抗体研究

梅毒是一种传染性很强的性传播疾病,早期诊断是防止其传播及治疗的关键。采用基因重组的梅毒螺旋体P47和P15抗原对289份临床标本进行梅毒螺旋体抗体的检测,并与常规方法进行了比较,结果表明：重组抗原ELISA法具有较高的敏感性和特异性,并能对梅毒进行早期诊断,可以代替常规方法检测梅毒螺旋体抗体。186份现患和已治愈梅毒患者标本,重组抗原ELISA法、TPHA法和RPR法均为阳性;60份健康献血员标本,重组抗原ELISA法和TPHA法、RPR法均为阴性;17份与梅毒患者有性接触者的标本,重组抗原ELISA法有2份阳性,而TPHA法、RPR法均为阴性,1个月后复查这2份血清TPHA和RPR均为阳性;6份RPR和类风湿因子均为阳性的血清,重组抗原ELISA法和TPHA法均为阴性,;20份肝硬化患者血清,3种方法检测均为阴性。     

梅毒实验室检测方法的研究进展

梅毒是由梅毒螺旋体引起的一种严重危害人类健康的性传播疾病,选择敏感性高、特异性强的检测方法对于梅毒的确证和临床指导应用均具有很重要的意义。当前,有多种梅毒实验室检测方法,其中直接检测法在近十年的临床诊断中仍被经常使用,血清学检测法仍然是目前梅毒实验室诊断的主要方法,以各类PCR技术为代表的梅毒分子生物学检测方法因其具有高度的特异性和敏感性已被广泛应用于临床检验。对梅毒实验室检测技术中直接法、血清学检测法及PCR检测法三类方法的研究进展进行了综述。     

化学发光法在梅毒实验诊断中的应用价值

目的:探讨化学发光法在梅毒实验诊断中的应用价值。方法:采用化学发光法、RPR法、TPPA法分别检测150例梅毒患者及125例非梅毒患者血清。结果:化学发光法、RPR法、TPPA法对150例梅毒血清标本和125例非梅毒血清标本对照组的敏感性分别为98.0%、75.3%和97.3%,特异性分别为98.3%、81.6%和97.5%。化学发光法、TPPA法敏感性和特异性明显高于RPR法,差异有统计学意义(P<0.05);化学发光法和TPPA法相比,敏感性和特异性差别不大,差异没有统计学意义(P>0.05);联合3种方法检测,梅毒诊断阳性率可提高到100%。结论:梅毒的化学发光检测法具有极高的敏感性和特异性,是一种自动化、定量检测方法,能够用于梅毒的准确诊断和疗效观察,与传统方法联检可防止误诊、漏诊,具有较大的临床应用价值。     

检测梅毒螺旋体抗体的纤维膜条方法的建立及其在诊断中的应用

目的：建立以纤维膜为载体的检测梅毒螺旋体抗体的方法,检查病人血清中对梅毒螺旋体多种抗原的抗体,用于梅毒感染的诊断。方法：将基因工程表达及纯化的梅毒螺旋体蛋白tp15、tp17、tp42和tp47分别结合在纤维膜上,用载抗原的纤维膜条检查血清中的抗体,抗体阳性者在相应抗原位置显示出特异条带。结果：梅毒螺旋体感染者血清中存在特异性抗体,在检查的460份临床诊断的患者血清中,对tp15、tp17、tp42和tp47抗原的抗体检出率分别为41.3%、100%、98.7%和51.7%;134份献血员血清抗体阴性。结论：建立的检测梅毒螺旋体感染的方法可同时检查对多种抗原的抗体,以纤维膜条作为诊断条检测血清抗体方法简便,用于临床诊断更特异、更敏感。     

梅毒螺旋体免疫相关膜蛋白的研究进展

梅毒是一种由梅毒螺旋体(Treponema.pallidum,Tp)感染所引起的慢性性传播疾病。近年来,其发病率居高不下,引起了全社会广泛的关注。随着分子生物技术的发展和人们的不断探究发现,膜蛋白可能在Tp致病过程中与宿主黏附、宿主免疫炎症反应等方面起着非常重要的作用,可能为Tp的主要致病因子。因此,对Tp膜蛋白的研究是认识其对宿主的致病性和进行致病机制研究的关键,就Tp的几种主要免疫相关膜蛋白的研究进展作了简要综述。     

Characterization of novel β‐1,3‐glucan recognition proteins from a Tibetan Plateau ghost moth Thitarodes pui (Lepidoptera,Hepialidae)

β‐1,3‐glucan recognition proteins (βGRPs) function as pattern recognition receptors in the innate immune response against invading pathogens. In the present study, we obtain full‐length cDNA clones for two novel putative βGRPs: TpβGRPc and TpβGRPd from the ghost moth Thitarodes pui (Lepidoptera: Hepialidae). Phylogenetic analysis shows a small distinct lineage, βGRP clade 4, consisting of T. pui βGRPs including TpβGRPa and TpβGRPb that have been identified previously. TpβGRPc and TpβGRPd, comprising 488 and 229 amino acids, have calculated molecular masses of 52 596 and 24 589 Da, respectively. TpβGRPc is 85.52% identical in sequence to TpβGRPa. TpβGRPb and TpβGRPd share the same deletion start site located at the conserved residue Pro 43, although TpβGRPd exhibits a much larger deletion of up to approximately 270 residues covering both the N‐ and C‐terminal regions. Affinity purification, associated with subsequent peptide sequencing, confirms the constitutive occurrence of TpβGRPa and TpβGRPc of similar size (approximately 65 kDa) in sixth‐instar larval haemolymph. These two βGRPs show clear binding affinities to curdlan, an insoluble β‐1,3‐glucan. A quantitative real‐time polymerase chain reaction analysis reveals the high‐level constitutive expression of TpβGRPc and TpβGRPd in the fat body of mid‐instar larvae, which are found to be susceptible to fungal pathogens in field investigations. Remarkable induction of both TpβGRPs occurs in response to haemocoelic challenge with entomopathogenic fungus Beauveria bassiana. The results of the present study suggest that TpβGRPs may contribute to the detection and control of fungal infections.     

Differential effect of testosterone and dihydrotestosterone on the sexual behavior of prepuberally castrated male rabbits

The effect of testosterone propionate (Tp) and dihydrotestosterone propionate (DHTp) at doses of 1, 3 and 9 mg daily for 30 days on the copulatory behavior of prepuberally castrated male New Zealand white rabbits was studied. Tp was significantly more effective than DHTp in eliciting copulatory behavior at each dose level tested. Three milligrams Tp was the minimal dose required to elicit mounting consistently. DHTp at the high dose level (9 mg) only elicited sexual activity comparable to that observed with the low dose of Tp (1 mg). The results suggest that T does not need to be reduced to DHT to stimulate sexual behavior in the male rabbit.     

Escape from CD8+ T cell response by natural variants of an immunodominant epitope from Theileria parva is predominantly due to loss of TCR recognition

Polymorphism of immunodominant CD8(+) T cell epitopes can facilitate escape from immune recognition of pathogens, leading to strain-specific immunity. In this study, we examined the TCR β-chain (TRB) diversity of the CD8(+) T cell responses of cattle against two immunodominant epitopes from Theileria parva (Tp1(214-224) and Tp2(49-59)) and investigated the role of TCR recognition and MHC binding in determining differential recognition of a series of natural variants of the highly polymorphic Tp2(49-59) epitope by CD8(+) T cell clones of defined TRB genotype. Our results show that both Tp1(214-224) and Tp2(49-59) elicited CD8(+) T cell responses using diverse TRB repertoires that showed a high level of stability following repeated pathogenic challenge over a 3-y period. Analysis of single-alanine substituted versions of the Tp2(49-59) peptide demonstrated that Tp2(49-59)-specific clonotypes had a broad range of fine specificities for the epitope. Despite this diversity, all natural variants exhibited partial or total escape from immune recognition, which was predominantly due to abrogation of TCR recognition, with mutation resulting in loss of the lysine residue at P8, playing a particularly dominant role in escape. The levels of heterozygosity in individual Tp2(49-59) residues correlated closely with loss of immune recognition, suggesting that immune selection has contributed to epitope polymorphism.     

How the Plant Temperature Links to the Air Temperature in the Desert Plant Artemisia ordosica

Plant temperature (Tp) is an important indicator of plant health. To determine the dynamics of plant temperature and self-cooling ability of the plant, we measured Tp in Artemisia ordosica in July, in the Mu Us Desert of Northwest China. Related factors were also monitored to investigate their effects on Tp, including environmental factors, such as air temperature (Ta), relative humidity, wind speed; and physiological factors, such as leaf water potential, sap flow, and water content. The results indicate that: 1) Tp generally changes in conjunction with Ta mainly, and varies with height and among the plant organs. Tp in the young branches is most constant, while it is the most sensitive in the leaves. 2) Correlations between Tp and environmental factors show that Tp is affected mainly by Ta. 3) The self-cooling ability of the plant was effective by midday, with Tp being lower than Ta. 4) Increasing sap flow and leaf water potential showed that transpiration formed part of the mechanism that supported self-cooling. Increased in water conductance and specific heat at midday may be additional factors that contribute to plant cooling ability. Therefore, our results confirmed plant self-cooling ability. The response to high temperatures is regulated by both transpiration speed and an increase in stem water conductance. This study provides quantitative data for plant management in terms of temperature control. Moreover, our findings will assist species selection with taking plant temperature as an index.     

The Tp38 (TpMglB-2) lipoprotein binds glucose in a manner consistent with receptor function in Treponema pallidum

A 38-kDa lipoprotein of Treponema pallidum (Tp38) was predicted to be a periplasmic sugar-binding protein based on its sequence similarity to the glucose/galactose-binding (MglB) protein of Escherichia coli (P. S. Becker, D. R. Akins, J. D. Radolf, and M. V. Norgard, Infect. Immun. 62:1381-1391, 1994). Inasmuch as glucose is believed to be the principal, if not sole, carbon and energy source for T. pallidum and is readily available to the spirochete during its obligate infection of humans, we hypothesized that Tp38 may serve as the organism's requisite glucose receptor. For the present study, a nonacylated recombinant form of Tp38 was coexpressed with GroES and GroEL in E. coli to facilitate the isolation of soluble, properly folded Tp38. The highly sensitive method of intrinsic fluorescence spectroscopy, predicated on the manner in which tryptophan residues reside and move within protein microenvironments, was then used to assess sugar binding to Tp38. The intrinsic fluorescence of Tp38 was essentially unaltered when it was exposed to D-mannose, D-fucose, D-ribose, L-glucose, or L-galactose, but it changed markedly in the presence of D-glucose, and to a lesser extent, D-galactose, indicating binding. The K(d) values for D-glucose and D-galactose binding to Tp38 were 152.2 +/- 20.73 nM and 251.2 +/- 55.25 nM, respectively. Site-directed mutagenesis of Trp-145, a residue postulated to contribute to the sugar-binding pocket in a manner akin to the essential Trp-183 in E. coli MglB, abolished Tp38's conformational change in response to D-glucose. The combined data are consistent with Tp38 serving as a glucose receptor for T. pallidum. These findings potentially have important implications for syphilis pathogenesis, particularly as they may pertain to glucose-mediated chemotactic responses by T. pallidum.     

梅毒螺旋体遗传物质和致病机制的研究进展

梅毒螺旋体(Treponema pallidum,Tp)是慢性全身性性传播疾病梅毒的病原体。由于Tp不能持续体外培养,阻碍了对Tp结构及其致病机制的深入研究。目前,Tp(Nicholes株)基因组测序的完成以及分子生物学技术的发展,为Tp的研究提供了机遇。就Tp的遗传物质和致病机制的研究进展进行综述。     

A novel beta-lactamase activity from a penicillin-binding protein of Treponema pallidum and why syphilis is still treatable with penicillin

Treponema pallidum, the causative agent of syphilis, is sensitive to penicillins. Yet, an abundant membrane-bound protein of this organism, Tp47, turns over penicillins. It is shown herein that the turnover process is a hydrolytic reaction that results in the corresponding penicilloates, products that have their beta-lactam bonds hydrolyzed. This is the reaction of beta-lactamases, bona fide resistance enzymes to beta-lactam antibiotics. Remarkably, the x-ray structure of Tp47 bears no resemblance to any other beta-lactamases or the related penicillin-binding proteins. Furthermore, evidence is presented that the reaction of Tp47 takes place in the absence of the zinc ion and does not involve intermediary acyl enzyme species. Hence, the beta-lactamase activity of Tp47 is the fifth known mechanism for turnover of beta-lactam antibiotics. Tp47 also exhibits a penicillin binding reaction, in the process of which the enzyme is covalently modified in the active site. The two reactions take place in two different active sites, and the events of the beta-lactamase activity are over 2,000-fold more rapid than the penicillin binding reaction. The level of beta-lactamase activity is high and is held back only by a strong product-inhibition component to the catalytic process. If natural selection would result in a mutant variant of Tp47 that overcomes product inhibition for the beta-lactamase activity, a novel bona fide resistance to penicillins will emerge in Treponema, which will be a disconcerting clinical development. The physiological functions of Tp47 are not known, but it is likely that this is at least a bifunctional enzyme involved in the processing of the Treponema peptidoglycan as a substrate.