布鲁氏菌病疫苗研究进展

使用疫苗免疫是防控布氏菌病(简称布病)的重要措施之一,但对人群使用疫苗免疫一直存有争议,世界上主要是俄罗斯(前苏联)和中国使用19-BA疫苗和104M疫苗对布病高危人群进行免疫.对于动物布病,早期曾使用灭活疫苗进行免疫,目前主要使用S19,Rev.1,RB51,S2和M5等弱毒活疫苗,其中Rev.1和S19是预防小反刍动物和奶牛布病最有效的疫苗,其它疫苗的使用也比较广泛.初步研究表明基因工程疫苗等新型布病疫苗具有良好的应用前景,但尚未被官方认可用于人和动物的布病免疫.特对人布病疫苗和动物布病疫苗进行介绍,并对基因工程疫苗以及其它新型布病疫苗的最新研究进展进行概述.     

布鲁氏菌病M5、S2疫苗免疫血清半抗原-琼脂扩散试验方法检测评价

目的 应用NH-AGID方法对布鲁氏菌M5、S2疫苗株免疫血清进行检测，评价该方法适用性。方法 选取3～5月龄羔羊74只，随机分为3组。分别以S2株1.0×10¹⁰ CFU、5.0×10⁹ CFU灌服和皮下注射接种羔羊各25只，M5株以1.0×10⁹ CFU皮下注射接种羔羊24只。免疫后7 d～150 d采集羊全血，分离血清，经RBT初筛和SAT确诊免疫抗体阳性血清。评估NH-AGID方法对M5和S2株疫苗免疫抗体与感染抗体的鉴别诊断特异性。结果 NH-AGID方法对M5株疫苗免疫阳性血清的LPS抗体检出率为100%,NH抗体检出率8.3%～29.2%,鉴别诊断特异性为82.8%。对S2株疫苗口服组免疫阳性血清的LPS抗体检出率为31.2%～66.7%,NH抗体检出率为0%;皮下注射组免疫阳性血清的LPS抗体检出率为45.4%～100%,NH抗体检出率为4.5%～20%。对S2株疫苗免疫血清的鉴别诊断特异性为96.5%。结论 NH-AGID方法不完全适用于羊用布鲁氏菌疫苗M5株和S2株的鉴别诊断，该方法的鉴别诊断特异性与...     

FPA与ELISA方法在我国部分地区牛布鲁菌病诊断应用中的比较研究

目的 评价荧光偏振试验(FPA)在我国部分地区临床牛血清样品检测中的应用。方法 荧光偏振试验(FPA)检测1 286份牛血清样本,其中布病感染阳性血清309份,阴性血清977份,ROC曲线分析FPA临界值;荧光偏振试验(FPA)、间接ELISA(IELISA)及竞争ELISA(CELISA)同时检测1 160份牛血清样本,其中布病感染阳性血清231份,阴性血清929份,Kappa分析检测方法一致性。结果 ROC曲线分析FPA最优临界值(cutoff)为95 mP,此时敏感性为100%,特异性为99.6%;比较FPA、IELISA、CELISA 3种方法,敏感性分别为97%、99.56%和98.27%,特异性分别为100%、98.06%和99.56%,同时3种方法一致性均≥0.75。结论 FPA检测技术可以用于动物布病诊断,在田间条件下,快速、准确诊断布病,避免动物二次抓捕,适于推广应用。     

牛种布鲁氏菌A19-△VirB12标记疫苗双重实时荧光定量PCR方-法的建立

目的 建立一种区分牛种布鲁氏菌A19-△VirB12标记疫苗株与布鲁氏菌野毒感染株的双重荧光定量PCR方-法。方法 分别以布鲁氏菌4型分泌系统中VirB8基因、VirB12基因序列设计2对引物及探针,优化实时荧光PCR反应体系及条件。以牛种布鲁氏菌A19-△VirB12标记疫苗株、牛种布鲁氏菌A19疫苗株、羊种布鲁氏菌M5疫苗株以及猪种布鲁氏菌S2疫苗株、大肠杆菌、沙门氏菌基因组DNA进行 Realtime-PCR扩增,评价该方-法特异性。分别构建布鲁氏菌VirB12基因和VirB8基因片段阳性质粒,10倍系列稀释后进行Realtime-PCR扩增,测定该方-法的敏感性。结果 本方-法具有良好的特异性,牛种布鲁氏菌A19疫苗株、羊种布鲁氏菌M5疫苗株以及猪种布鲁氏菌S2疫苗株基因组DNA同时出现VirB8基因与VirB12基因阳性扩增,牛种布鲁氏菌A19-△VirB12标记疫苗株仅出现VirB8基因阳性扩增,大肠杆菌、沙门氏菌均未扩增出目的条带,对VirB8基因及VirB12基因片段阳性质粒的检测限分别为约10² copies/μL和10³ copies/μL。该方-法仅用于鉴别区分牛种布鲁氏菌A19-△VirB12标记疫苗株与布鲁氏菌野毒株。结论 本研究建立的布鲁氏菌双重Realtime-PCR方-法,具有良好的特异性和敏感性,为今后鉴别牛种布鲁氏菌A19-△VirB12分子标记疫苗免疫牛与自然感染牛提供技术支撑。     

布鲁氏菌重要膜蛋白研究进展

布病是由布鲁氏菌属细菌引起的一种重要的人兽共患病,在世界范围内广泛分布。布病不仅可导致巨大的经济损失,也是威胁人群健康的主要风险因素。布鲁氏菌外膜蛋白是主要的免疫和保护性抗原,不仅与布鲁氏菌的毒力和细胞内生存有密切的关联,而且对开发和建立新型的特异性血清学诊断方法具有重要的意义。本文对布鲁氏菌的重要外膜蛋白的研究进展予以综述,从而更好的理解布鲁氏菌表面蛋白的抗原特性,为建立布病实验室诊断方法和新型疫苗研发提供参考。     

酶联免疫吸附试验(ELISA)诊断慢性布鲁氏菌病价值的探讨

本文对 ELISA 诊断慢性布鲁氏菌病的价值进行了分析,通过与 ISAT 和 Coomb's试验比较,证明其敏感性最好,特异性也很好。ELISA 能直接地定量测定血清中特异的IgG 和 IgM 抗体。慢性布鲁氏菌病和因错误接种布鲁氏菌菌苗而发病的患者血清中的抗体类型主要是 IgG。而予防接种者主要是 IgM,且它在血清存在时间较短。因而为解决感染与免疫的鉴别提供了线索。     

布鲁氏菌病自然感染与菌苗免疫鉴别诊断研究进展

为防制动物布鲁氏菌病(以下简称布病),成功地研制出了布鲁氏菌(以下简称布氏菌)羊种M_5弱毒疫苗和猪种S_2弱毒疫菌等,并进行了大面积的推广应用,对牛、羊、猪、鹿等动物布病的防制起了具大的作用,经济效益和社会效益十分显著,但是存在的问题是布氏菌自然感染和菌苗免疫的鉴别,因为人畜应用活菌苗以后所产生的各种血清学反应和自然感染所产生的各种血清学反应极其相似。鉴别诊断不仅关系着布病的防治工作,同时对流行病学调查也具有重要意义,这也是布病防治领域的难题之一。多年来,国内外学者为解决这一具有战略意义的课题,在建立不同种类的检测方法,分析布氏菌免疫和感染的机体体液中的不同类型免疫球蛋白抗体出现的时间、在数量上的差别和动态上的关系,以及制备和纯化各种布氏菌的抗原等方面进行了广泛深入的研究。     

用布鲁氏菌膜蛋白抗原的免疫酶斑点试验检测布鲁氏菌病患者抗体

本文报告用布鲁氏菌外膜蛋白抗原。对142份布鲁氏菌病急,慢性患者血清进行免疫酶斑点试验,为减少样品的交叉凝集、省试剂和携带方便,又将纤维素膜用打孔器打成小圆片备用。结果表明,就敏感性、特异性和操作程序而言,免疫酶斑点试验要优于ELISA,可供大量布病血清检查之用。所采用的外膜蛋白抗原,将为今后诊断粗糙型及非典型布鲁氏菌引起的布鲁氏菌病提供了广谱抗原。     

布鲁氏菌抗体胶体金免疫层析法快速检测技术的建立

目的建立一种快速检测布鲁氏菌抗体的新方法。方法利用胶体金免疫层析技术,以大肠埃希菌表达、纯化的OMP31与BP26重组蛋白作为检测抗原,以金黄葡萄球菌A蛋白(SPA)作为胶体金标记物,制备胶体金免疫层析试纸条,用于检测布鲁氏菌抗体,并分析方法的敏感性和特异性。结果以粒径为40nm胶体金制备的试纸条检测布鲁氏菌抗体的敏感性最高,胶体金最佳标记pH为6.2,SPA蛋白最适标记量为6μg/ml。交叉试验证明试纸条不与其他非相关疾病感染血清反应,特异性高。试纸条检测结果与琥红平板试验方法的符合率为92%。结论制备的布鲁氏菌抗体胶体金免疫层析试纸条具有敏感、特异、简便、快速的特点,可用于鉴别布鲁氏菌自然感染和人工免疫,并可区分牛、羊种布鲁氏菌感染,可在基层推广使用。     

滴金免疫测定法检测不同人群布鲁氏菌病

目的探讨滴金免疫测定法在不同人群中检测布鲁氏菌感染的应用价值。方法用已建立的布病滴金免疫测定技术检测不同职业和不同感染类型人员的布病抗体,并与布病试管凝集试验作平行检测。结果滴金免疫测定法与试管凝集试验两种方法符合率为99.88%,阳性符合率达到94.25%;且本法对布病发病人群和高滴度血清样本具有更高的检测敏感性;对不同职业人群检测,接触山羊和奶牛职业人群的布鲁氏菌病抗体检出率分别为6.40%和1.32%。结论滴金免疫测定法对布病疫区人群和非疫区重点职业人员开展布病监测具有实用价值,也可用于布病的临床检测和流行病学筛查。     

结核分枝杆菌特异性蛋白抗体检测在结核病诊断中的价值

目的研究结核分枝杆菌特异性蛋白（TB-SA）抗体检测在结核病诊断中的价值。方法对829例结核病患者（男471例，女358例）、278例非结核性肺部疾病患者（男172例，女106例）和125例健康志愿者（男51例，女74例）同时进行结核菌素纯蛋白衍生物（PPD）试验，结核分枝杆菌TB-SA抗体检测，并对全部肺结核和非结核性肺部疾病患者进行痰抗酸杆菌涂片和培养。采用EPl2000统计软件进行统计学分析，组间比较采用t检验，计数资料采用X^2检验。结果结核分枝杆菌TB-SA抗体检测诊断菌阳肺结核的敏感度为75．1％（272／362）；诊断菌阴肺结核的敏感度为68．9％（226／328）；诊断肺外结核病的敏感度为71，2％（99／139）；诊断结核病的总体敏感度为72．0％（597／829），特异度为82．1％（331／403）。TB-SA血清抗体检测值并不与PPD值呈线性关系，即结核分枝杆菌TB-SA抗体检测诊断结核病不受卡介苗接种反应的影响。结论结核分枝杆菌TB-SA抗体检测诊断结核病有较好的敏感度和特异度，是辅助诊断和鉴别诊断结核病的有效手段。     

Gelatin particle agglutination (PA) antibody: comparison to neutralizing antibody (NT) and hemagglutination inhibition (HI) antibody and relationship to IgG avidity

Changes in gelatin particle agglutination (PA), hemagglutination inhibition (HI), and neutralization (NT) antibodies were compared using sera from 124 individuals collected between 3 weeks and 10 years after measles vaccination, and the relationship between these changes and IgG avidity was studied. PA, HI, and NT antibodies peaked 4-5 months after vaccination. The rate of increase in mean antibody titer from 0-1 months after vaccination to peak levels was 1.7-fold for NT, 1.5-fold for HI, and 7.4-fold for PA antibodies. Peak mean antibody titer was 2(11.8) for PA, 2(6.7) for NT and 2(6.7) for HI antibodies. After peaking PA antibodies changed in parallel with NT and HI antibody titers, and correlated strongly with both antibodies (r = 0.801 and 0.840). In contrast, NT and HI antibodies were consistent throughout the period. IgG avidity increased for 4-5 months following vaccination, peaking at 45%, and remaining constant at 40-50% for the next 10 years. PA antibody is strongly influenced by IgG avidity, unlike NT and HI antibodies. Due to the effects of IgG avidity, PA antibodies increase more significantly than NT and HI antibodies as IgG antibodies mature following vaccination, resulting in a weak correlation between PA and NT or HI antibodies. Following the increase in IgG avidity to maturation, PA antibodies correlated strongly with NT and HI antibodies. PA assay detected IgM antibodies against measles virus more efficiently than the NT test. The PA assay thus differs from conventional, commonly used NT and HI assays. PA assay is simple and rapid, making it very useful for detecting measles antibodies provided that its unique features are taken into accounts.     

Detection of IgM antibody against region IV flagellin of Salmonella paratyphi A

Salmonella paratyphi A is a pathogenic bacterium that causes paratyphoid fever. The current laboratory diagnostic techniques are unsatisfactory. To improve diagnosis, a plasmid (pSK-8E) encoding phase 1 flagellin gene nucleotide position 452-890 from S. paratyphi A has been constructed. The recombinant protein expressed from the plasmid has been used to develop an indirect ELISA for IgM antibody detection. Sera from patients with hemoculture positive for S. paratyphi A, S. typhi, other gram-positive and gram-negative bacteria, and dengue hemorrhagic fever as well as from healthy control subjects were tested. Sensitivity, specificity, positive and negative predictive values of the test were 56.9%, 98.8%, 90.6% and 92.1%, respectively. Since the sensitivity was low, the explanation for this result was investigated. It was found that the sensitivity of the test could be increased to 83.3% if the sera were obtained 9-12 days after onset of fever. The sera obtained earlier or later gave only 33.3% and 66.6% sensitivity, respectively. This result suggests that the IgM antibody detection assay which we have developed is a valuable tool for diagnosis of S. paratyphi A infection when the serum samples are taken at the appropriate time.     

斑点金免疫渗滤法快速诊断布鲁杆菌病的研究

目的建立一种布鲁杆菌病快速免疫诊断方法。方法以硝酸纤维素膜为载体,胶体金标记蛋白为显示剂,通过最佳实验条件的选择及稳定性、重现性、敏感性、特异性和交叉反应试验,建立斑点金免疫渗滤法(DIGFA)快速检测布鲁杆菌病。结果 DIGFA快速检测布鲁杆菌病具有很好的重现性和稳定性;敏感性、特异性分别达到98％和100％;未发现与肺结核患者、乙肝患者及幽门螺杆菌感染者血清有交叉反应。结论 DIGFA快速检测布鲁杆菌病快速、简便,测试过程1-2 min内完成,试剂敏感、特异、稳定,非常适合布鲁杆菌病的诊断及流行病学调查。     

结核分枝杆菌特异性蛋白抗体检测在肺外结核病诊断中的价值

目的研究结核分枝杆菌特异性蛋白(TB-SA)抗体检测在肺外结核病诊断中的价值。方法对71例肺外结核病患者和40例健康志愿者进行血清结核分枝杆菌特异蛋白抗体检测,并对各种肺外结核病引流物标本进行AFB涂片和培养检查。结果结核分枝杆菌TB-SA抗体检测诊断菌阳肺外结核病敏感度为87.5%(7/8),诊断菌阴肺外结核的敏感度为63.5%(40/63),诊断肺外结核总体敏感度为66.2%(47/71),特异度为97.5%(39/40)。结论结核分枝杆菌TB-SA抗体检测诊断肺外结核病有较好的敏感性和特异性,是辅助诊断和鉴别诊断肺外结核病较为理想的免疫学方法之一。     

Practicability and reliability of a new rapid detection kit for rubella antibody

We have investigated the practicability and reliability of a new rapid detection kit for rubella antibody by means of an immunochromatographic assay. This kit can measure 100 microliters of total blood or 50 microliters of serum or plasma in approximately 10 minutes. When a band appears in this kit, a sample is positive. The subjects of this investigation were 233 medical students or nursing students. Blood was also drawn from their fingertips of 71 of them. The blood was obtained with a lancet for measuring blood sugar. By an ELISA assay using IgG antibody for rubella, 188 samples (80.7%) were found to be positive, 1 sample (0.4%) was +/-, and 44 samples (18.9%) were negative. The positive IgG antibody titers deviated toward the lower levels since the median was lower than the mean. Therefore, the subjects were appropritate for evaluation of low titer samples. In comparison with the measurement of IgG antibody using the ELISA assay, the sensitivity and specificity levels of this new kit were 99.5% and 100%, respectively. The correlation index between the antibody titers and color concentration of the band was as weak as 0.5 (0.35-0.58 at 95% confident range). Seroconversion of the antibody in paired samples is necessary for diagnosis of rubella in this kit. However, if there is an uncertain past history of vaccination and/or rubella, this kit is useful for evaluating the need for vaccination in 10 minutes. It usually takes approximately one week to receive antibody results through a commercial laboratory.     

免疫荧光法检测炭疽荚膜抗体早期诊断炭疽病的探讨

炭疽病人发病3天后,开始产生抗炭疽荚膜抗体,可用免疫荧光法测出。发病4～7天同阳性率增至68.2％,一周后患者大多呈阳性。由于特异性的炭疽荚膜抗体只产生于炭疽显性感染患者体内,故在临床上可用于作炭疽病的早期快速诊断和鉴别诊断以及流行病学的追索诊断,本文结果比较满意。该法准确、快速、简易,值得推广。     

布鲁氏菌OMP25与OMP31蛋白的表达及其间接ELISA诊断试剂盒研制

目的 为建立布鲁氏杆菌间接ELISA检测方法。方法 本研究根据GenBank中已登录猪种布鲁氏菌S2株全基因组序列,分别针对omp25和omp31设计一对引物,分别扩增并回收573 bp与783 bp目的基因片段,将其分别克隆入pET-28a原核表达载体中,构建重组质粒pET-OMP25与pET-OMP31,进行酶切鉴定与基因序列测定后,转化BL21表达菌,经IPTG诱导表达,OMP25与OMP31蛋白主要以包涵体形式存在;将目的蛋白纯化,应用Western blot进行蛋白活性的检测。结果 纯化的目的蛋白具有良好的免疫学活性。以纯化的重组蛋白为包被抗原,在确定了OMP25与OMP31蛋白最佳配比为4:1、抗原最佳包被浓度为10 μg/mL、血清的最佳稀释倍数为1:50倍稀释、临界值为0.314的基础上,建立羊布鲁氏菌病抗体间接ELISA诊断方法并组装成诊断试剂盒,并将试剂盒进行了特异性、敏感性、重复性、符合率试验、血清交叉反应性等检测。结论 研制的羊布鲁氏菌病抗体ELISA诊断试剂盒具有良好的特异性、敏感性,可重复性,可应用于临床样本检测。     

Specific antibody responses to West Nile virus infections in horses preimmunized with inactivated Japanese encephalitis vaccine: evaluation of blocking enzyme-linked immunosorbent assay and complement-dependent cytotoxicity assay

West Nile virus (WNV) and Japanese encephalitis (JE) virus are distributed separately in the world with some exceptions. There is a concern that WNV may invade into Asia where JE virus exists. On and after such invasion, any differential diagnosis could be complicated by serological crossreactivities. We previously demonstrated experimentally using horses infected with WNV that preimmunization with inactivated JE vaccine considerably affected the ability of neutralization tests and immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (ELISA) to diagnose WNV infection. Here, we investigated WNV-specific antibody responses in vaccinated horses using a blocking ELISA and complement-dependent cytotoxicity (CDC) assay to evaluate these two newly developed serodiagnostic methods for WNV infection. Sera previously collected from six experimentally infected horses were used: Three were vaccinated before the infection, whereas the other three remained unvaccinated. WNV-specific antibody responses were successfully detected in the vaccinated and unvaccinated horses using both new methods, except for one vaccinated horse in which responses were not induced, probably as a result of crossprotection induced by JE vaccination. Specific antibody responses were at earliest detected from days 9 to 10 postinfection in the blocking ELISA, whereas the CDC assay provided earlier detection (at days 7-8) in all horses. The time courses of antibody levels were similar between vaccinated and unvaccinated horses in either method, indicating no notable effect of vaccination on detection of specific antibody responses, as far as antibodies were induced. These results indicated that blocking ELISA, but preferably the CDC assay, can be useful for detecting WNV infection in JE-vaccinated horses.     

抗环瓜氨酸多肽抗体检测早期诊断类风湿关节炎研究

目的探讨抗环瓜氨酸多肽(CCP)抗体检测对类风湿关节炎(RA)早期诊断的意义。方法应用ELISA法检测2004—2005年中国医科大学附属盛京医院150份人血清的抗CCP抗体,包括54例RA患者,80例其它风湿病患者,16名正常人;并分析抗CCP抗体与类风湿因子(RF)、C反应蛋白(CRP)、血沉(ESR)的相关性。结果抗CCP抗体对RA的敏感性和特异性分别为70·4%和93·8%。发病2年内与2年以上的抗CCP抗体阳性率差异无显著性。抗CCP抗体阴性组与阳性组的关节畸形率差异无显著性。抗CCP抗体与RF、CRP、ESR无相关性。结论抗CCP抗体对RA具有较好的敏感性和很高的特异性,联合抗CCP抗体和RF可以提高诊断的准确性,对RA的早期诊断具有重要意义。