检测血清幽门螺杆菌细胞空泡毒素抗体的间接ELISA法初步建立

目的:以重组细胞空泡毒素(VacA)蛋白为抗原初步建立检测血清VacA抗体间接ELISA法,为制备检测Hp毒株感染的试剂盒奠定基础.方法:Ni-NTA2 树脂纯化重组VacA蛋白,用重组蛋白免疫兔,Helico Blot试剂盒检测兔血清VacA抗体以鉴定其免疫原性.Western blot检测其抗原性.并以纯化的重组蛋白为抗原,建立检测血清VacA抗体间接ELISA法,以Helico blot试剂盒为标准,确定该方法的特异性与敏感性.结果:以重组蛋白为抗原建立的检测血清VacA抗体的间接ELISA法,敏感性与特异性分别是93.1%和92.5%.结论:以重组蛋白为抗原初步建立检测血清VacA抗体的间接ELISA法,敏感性、特异性高,为制备商品化的试剂盒奠定了基础.     

基于可溶性虫卵抗原的血吸虫抗体检测免疫传感器

目的:将免疫法特异性和电化学法灵敏性相结合,建立一种高灵敏基于可溶性虫卵抗原的电化学免疫传感器,用于血吸虫抗体的检测。方法:巯基乙胺通过吸附作用在金电极表面形成的自组装单分子层(SAM),依次滴加2.5%戊二醛和血吸虫抗原(可溶性虫卵抗原,soluble egg antigen,SEA),用以捕获抗SEA抗体(兔多抗或人多抗),然后再与辣根过氧化物酶(HRP)标记的二抗形成免疫复合物。用循环伏安法检测还原峰的电流值。结果:在底物为添加了2μl 30%的双氧水及1.0 mmol/L对苯二酚的0.1 mol/L pH7.2的PBS缓冲液中,还原峰的电流值与抗SEA兔血清稀释度在10-3与10-6范围内成正比,相关系数为0.956。与抗SEA人血清稀释比在10-1与10-4范围内成正比,相关系数为0.988。在1∶40稀释度条件下,血吸虫病人与正常人血清的抗SEA还原峰的电流值相差7.4μA。结论:该方法可以用于血吸虫抗体的初步检测。     

弓形虫SAG1重组蛋白的制备及血清学检测应用

目的:以重组弓形虫表面抗原SAG1对上海市徐汇区人群进行弓形虫抗体血清阳性率分析。方法:重组质粒pET-19b-SAG1在大肠杆菌BL21star(DE3)pLysS中诱导表达,经Ni2+亲和层析法纯化目的蛋白后,用ELISA鉴定其抗原特异性。以SAG1重组蛋白为抗原用ELISA方法检测上海市徐汇区人群血清(266份)弓形虫IgG抗体,并与国外试剂盒进行比较。结果:SAG1重组蛋白以包涵体形式存在,产量为0.98 mg/100 ml培养基,纯度达95%以上,并具有较好的抗原特异性。以该重组抗原对上海市徐汇区人群弓形虫血清抗体进行IgG-ELISA检测,显示阳性率为9.4%(25/266)。结论:成功表达提纯了具有较高抗原特异性的SAG1重组蛋白。本次调查上海市徐汇区人群的弓形虫血清抗体阳性率为9.4%,建议加强血清学监测,便于及时采取有效干预措施。     

抗烟曲霉单克隆抗体用于侵袭性烟曲霉感染病原学检测的实验研究

目的探讨自行研制的抗烟曲霉单克隆抗体在兔侵袭性烟曲霉感染动物模型中的应用。方法建立兔侵袭性烟曲霉感染动物模型;用双抗体夹心酶联免疫吸附试验法(ELISA)检测兔血清中烟曲霉抗原;以免疫酶组织化学方法检测兔肺、肝、脾、肾组织中烟曲霉抗原。结果血清中烟曲霉抗原检测:接种烟曲霉抗原后24、48、72、96 h,血清中烟曲霉抗原检测阳性;免疫酶组织化学检测:接种后96 h,肺、肝、脾、肾组织免疫酶组织化学染色均为阳性。结论抗烟曲霉单克隆抗体在侵袭性烟曲霉感染的病原学诊断中有较大的应用前景。     

应用重组致密颗粒抗原6检测诊断弓形虫病的研究

目的以纯化的重组致密颗粒抗原6作为检测抗原,建立检测弓形虫IgM和IgG抗体的ELISA新方法,为临床治疗提供参考依据。方法 2007年1月-2011年12月对59份弓形虫阳性血清标本进行检测,并与进口弓形虫ELISA-IgM和IgG试剂盒进行比较,数据采用SPSS 13.0软件进行统计分析。结果 ELISA法优化检测条件为包被抗原浓度为40μg/ml;敏感度比较表明血清稀释度在1∶10~1∶80为优;特异性试验表明IgM阳性的抑制率为97.36%、IgG阳性的抑制率为97.98%;用rGRA6-IgM-ELISA对混合弓形虫IgM阳性和阴性血清的精密度检测表明,IgM阳性的变异系数(CV值)为2.76%,IgM阴性混合血清的CV值为0.45%;用rGRA6-IgG-ELISA对混合弓形虫IgG阳性和阴性血清的精密度检测表明,IgG阳性的变异系数(CV值)为2.89%,IgG阴性混合血清的CV值为1.65%;rGRA6-IgM-ELISA与进口试剂盒的总符合率为91.01%,rGRA6-IgG-ELISA与进口试剂盒的总符合率为94.59%。结论重组抗原rGRA6能被弓形虫感染患者血清IgM和IgG抗体所识别,用重组抗原rGRA6构建的试剂盒诊断弓形虫病具有较高的特异性、敏感性。     

免疫酶染色试验诊断弓形虫感染的初步研究

以弓形虫的玻片为抗原，进行了免疫酶染色试验（ＩＥＳＴ），检测２０份阳性兔血清和５２份阳性人血清，阳性率分别为１００％和９６．１５％；２０份阴性兔血清和１９３份阴性人血清，假阳性率为０和１．０４％；此法与血吸虫病、肝吸虫病及疟疾均无交叉反应，说明对弓形虫有较高的特异性；用该法对精神病患者、孕妇及健康人群进行了弓形虫感染的检测，其结果分别为２４．５１％、１３．３５％和７．３２％，此结果与染色试验（ＤＴ）无显著性差异。ＩＥＳＴ法具有简便、经济、敏感、特异等优点，适用于现场推广应用。     

感染日本血吸虫兔粪便中的抗原物质

花村·阿久沢把感染日本血吸虫兔的粪便抽出液作检材,与抗日本血吸虫兔血清进行凝胶内沉淀反应,证实了抗原物质的存在(久留米医会志,34,886～888,1971)。这次把抗原物质通过凝胶过滤层析,用 Ouchterlony 法研究了各层与抗日本血吸虫兔血清反应,进行了免疫活性分层的探讨。其方法如下:取粪便50克,加入三倍量的磷酸缓冲液(pH7.2)一起将其搅碎,纱布过滤,把滤液以12,000转/分的转速离心30分钟,取上清液作为粪便抽出液。凝胶过滤是用葡聚糖 G—50和 G—200,在1/50M 磷酸缓冲液及水中进     

江苏预防医学第17卷总目次

论著江苏省部分城市男男性接触者行为学特征分布羊海涛丁建平陈国红等1.1………………………………重组抗原金标免疫渗滤法检测弓形虫感染的研究侯敏褚宏亮芦王英等1.4………………………………江苏省城乡居民糖尿病流行特征及相关因素研究潘晓群袁宝君杭万双等1.6………………     

胶体金免疫层析法检测弓形虫循环抗原的研究

目的：建立一种简易快速、自测式胶体金免疫层析法（GICA）用于检测血清中弓形虫循环抗原（CAg）。方法：用胶体金颗粒标记弓形虫单克隆抗体，制成免疫层析测试条。血清中的弓形虫循环抗原与测试条上的金标记单克隆抗体结合后沿着硝酸纤维素膜移动，与膜上的固相抗体结合形成肉眼可见的红色线条。结果：用GICA与ELISA比较检测105份小鼠血清中循环抗原，两法符合率为95％（P〉0．05）。结论：GICA检测血清中的弓形虫循环抗原特异性强、灵敏度高、简便快速，无需特殊仪器设备，有广泛应用价值。     

日本血吸虫童虫早期诊断抗原模拟表位的初步筛选研究

目的：利用噬菌体随机12肽库技术筛选日本血吸虫感染早期诊断抗原。方法：用日本血吸虫感染后21d兔血清从噬菌体12肽库中经3轮筛选,有效地富集与早期感染兔血清有亲和力的噬菌体克隆。随机挑取11个单克隆分别纯化、扩增,随后采用ELISA、DOT-ELISA等检测方法,挑选出与早期感染兔血清有高亲和力的噬菌体克隆。结果：挑选出3个与感染后21d兔血清具有高亲和力的噬菌体单克隆。结论：用感染后21d兔血清从噬菌体随机12肽库中能筛选到日本血吸虫童虫早期诊断抗原模拟表位。     

Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody

Background:

Cystic echinococcosis is an important zoonosis caused by the Echinococcus granulosus with a substantial impact in human and animal health in endemic areas. The purpose of the present study was serodiagnosis optimizing of dog echinococcosis in order to achieve a rapid diagnostic method.

Methods:

Eight dogs were challenged with protoscoleces in order to have positive echinococcosis serum and 2 two-month old puppies were used as uninfected controls. Colloidal gold was prepared by controlled reduction of a boiling solution of chloroauric acid (H [AuCl4]) with sodium citrate and labeled with recombinant EPC1. Dot immunogold filtration assay (DIGFA) was developed by coating rEPC1 labeled colloidal gold on nitrocellulose membrane. The canine sera, taken three times including, 15, 28 and 35 days post infection were tested. A total of 30 serum samples including 24 sera from 8 infected dogs and 6 sera from 2 puppies were comparatively detected with both DIGFA and ELISA.

Results:

Gold labeled antigen, showed a dark purple dot with agglutination particles in positive sera and light purple dot without agglutination in negative sera. Among 30 serum samples, 23 were positive and 7 were negative with DIGFA and 24 were positive, 6 were negative with ELISA.

Conclusion:

DIGFA as a rapid and simple procedure could be utilized in quickly diagnosis of echinococcosis.     

弓形虫PCR试剂盒对感染家兔血液和精液的检测

目的：评价国内公司研制的弓形虫PCR检测试剂盒。方法：采用市售的国内生产的4种弓形虫PCR试剂盒检测弓形虫阳性兔的血液及精液标本。结果：温州伊利康TOX-PCR试剂盒、北京美迪科TOX-PCR试剂盒对家兔感染前后血液、精液标本均未出现阳性结果。上海复生TOX-PCR试剂盒检测家兔精液标本感染前阴性，感染后均能出现相应的结果，而检测血液标本杂带太多。洛阳华美TOX-PCR试剂盒检测家兔血液标本、精液感染前均为阴性，感染后血液标本阳性率76．53％，43份精液标本阳性率2．33％。结论：目前国内市售的TOX-PCR试剂盒可检测各种组织标本，但实际应用价值局限。     

问号钩端螺旋体属特异性LipL41s抗原膜定位及其患者血清抗体检测

目的确定问号钩端螺旋体（钩体）属特异性脂蛋白抗原LipL41s膜定位及其自然抗体应答情况和抗体类型。方法采用显微镜凝集试验（MAT）检测四川地区钩体病患者血清标本。IPTG诱导重组原核系统表达目的蛋白rLipL41／1和rLipL41／2，Ni—NTA亲和层析法提纯目的重组表达产物。采用Westernblot检测感染不同血清群问号钩体病患者血清与rLipL41s的免疫反应性。采用胶体金免疫电镜技术，对LipL41s进行膜定位。建立基于rLipL41s的ELISA，检测钩体病患者血清中特异性抗体水平及其类型。结果黄疸出血群是四川地区最主要的优势钩体血清群。不同血清群问号钩体病患者血清均能有效识别LipL41s。LipL41s是位于钩体外膜表面的蛋白分子。156例MAT阳性钩体病患者血清标本中，rLipL41／1和rLipL41／2特异性IgM阳性率分别为84．6％～87．8％和78．2％～83．3％，特异性IgG阳性率分别为69．2％～81．4％和75．0％～80．1％。结论LipL41s是钩体表面蛋白抗原。自然感染钩体时，LipL41／1和LipL41／2可诱导机体产生IgM和IgG两类血清抗体，且两者之间有广泛的抗原交叉反应。rLipL41／1和rLipL41／2可作为研制通用型钩体基因工程疫苗和检测试剂盒的候选抗原。     

Enzyme-linked immunosorbent assays for the measurement of specific antibodies in experimentally induced ovine toxoplasmosis

Tachyzoites of the RH strain of Toxoplasma gondii were inoculated intravenously into sheep following which serum samples were collected at approximately weekly intervals for 9 months. The sera were examined by the toxoplasma dye test and two enzyme-linked immunosorbent assays (ELISA) specifically developed for investigations of ovine toxoplasmosis. One was an antibody class capture assay for the detection of anti-toxoplasma specific IgM, the other an indirect assay which detected anti-toxoplasma IgG. Some of the sheep had antibodies to toxoplasma prior to inoculation but none had specific IgM. Sera collected 17 days after inoculation showed that all had raised specific antibody levels but the only sheep that produced specific antitoxoplasma IgM were those that were initially without any antibody. Specific IgM could be detected in all these particular sheep for at least 1 month after infection and up to 3 months in some. Specific IgG persisted at high levels for at least 3 months and could still be detected at moderate levels for at least 9 months. The ELISA methods described are simple to perform and could clearly distinguish between previous infection and this experimental infection with Toxoplasma gondii.     

New method of serological testing for Mycobacterium avium subsp. paratuberculosis (Johne's disease) by flow cytometry

Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Attempts to control JD have proven inordinately difficult due to low levels of sensitivity by currently available diagnostic tests, which are also incapable of detecting prepatent MAP infections. In the present work, we describe the use of a flow cytometry method (FCM) for serological diagnosis of subclinical and clinical JD in cattle. The FCM was capable of distinguishing MAP-infected from MAP-non-infected cattle as well as MAP from M. scrofulaceum and M. avium subsp. avium. Results of the FCM were compared to that of a commercially available ELISA using 82 serum samples from JD-positive and JD-negative dairy and beef cattle farms that were separated into the following groups: (1) sera from a JD-free farm; (2) sera from JD-positive farms that had tested negative by ELISA; and (3) sera from JD-positive farms that tested JD-positive by ELISA. The FCM found that groups 1-3 were 6.6%, 73.3%, and 97.3% positive for MAP infections, respectively. By using 30 fecal culture-negative samples from a JD-free farm and 21 fecal culture-positive samples from JD-positive farms, diagnostic sensitivity and specificity of the FCM were calculated to be 95.2% and 96.7%, respectively. A retrospective study of 10 JD-positive cows showed that the FCM detected MAP infections 6-44 months earlier than the fecal culture test. Further, the FCM specifically detected MAP infections in serum samples as early as 170 days after experimental inoculation of calves with MAP and did not react with calves inoculated with other mycobacteria. Production of IgG against MAP was detected by FCM in all the calves inoculated with MAP 240 days after inoculation, whereas positive anti-MAP IgG production was not detected in control calves or calves experimentally infected with M. avium subsp. avium or M. bovis. The FCM assay is rapid and is completed in less than 4 h. Moreover, the FCM is objective, technically easy and can be automated for handling large numbers of samples. This novel assay might form the basis of a highly sensitive and subspecies-specific test for the diagnosis of JD.     

孕妇血清中抗弓形虫抗体IgG和IgM检测分析

目的分析孕妇感染弓形虫的规律变化情况。方法采取酶联免疫法对2009至2011年间到本院进行孕检的3176例孕妇进行血清中抗弓形虫抗体IgG和IgM的检测,对比不同年份、不同季节、不同动物接触史孕妇的感染阳性率。结果第一季度、第二季度弓形虫感染率显著高于第三、第四季度,P〈0.01;有动物接触史孕妇的感染率显著高于无动物接触史的孕妇,P〈0.01。结论于孕早期进行弓形虫抗体IgG与IgM检测可尽早发现弓形虫,及时进行相应治疗,有效提高新生儿健康水平。     

胶体金免疫层析法快速诊断布氏菌病的研究

目的 研发一种布氏菌病快速诊断试剂.方法 用胶体金免疫层析法(GICA)试剂测试布氏菌病患者和健康人血清的敏感性、特异性、交叉反应、重现性和稳定性;并与DIGFA及SAT比较.结果 GICA试剂敏感性和特异性分别为93.6%和94.9%,youden指数为O.885.检测结果与DIGFA及SAT相比,三者差异无统计学意义(P>0.05),本法未发现与肺结核患者、乙型肝炎患者及幽门螺杆菌感染者血清有交叉反应,有很好的重现性和稳定性.结论 GICA具有试剂敏感、特异和稳定;方法简便、快速和微量的特点,适合于布氏菌病的诊断、流行病学调查及现场应用.     

外周血单个核细胞感染乙型肝炎病毒对新生儿宫内感染的影响

目的 :探讨 HBs Ag、 HBe Ag阳性孕妇外周血单个核细胞 ( PBMC)内乙型肝炎病毒 ( HBV) DNA感染状况及其在宫内母婴垂直传播中的作用。方法 :对 HBs Ag/ HBe Ag双阳性共 6 7对孕妇及其新生儿静脉血分离和提纯 PBMC后 ,经抽提、纯化后的 DNA进入 PCR扩增反应 ,引物为 HBV C区基因序列。结果 :6 7例 HBs Ag及 HBe Ag双阳性的孕妇中有 35例 ( 5 2 .2 % )PBMC中 HBV DNA阳性 ,2 5例孕妇在血清及 PBMC中均发现 HBV DNA。6 7例新生儿有 2 2例感染 HBV DNA,感染率 32 .8% ,其中血清 HBV DNA阳性者 10例 ,PBMC HBV DNA阳性者 19例 ,二者均阳性者 7例。结论 :母亲 PBMC内 HBV DNA阳性可能导致新生儿 PBMC中 HBV DNA阳性 ,PBMC内的 HBV DNA可能是 HBV母婴垂直传播的一条重要途径 ,同时 ,HBs Ag及HBe Ag阳性母亲若血清 HBV DNA为阳性就极大增加了其新生儿感染 HBV的危险性     

An enzyme-linked immunosorbent assay for diagnostic detection of Taenia saginata copro-antigens in humans.

An immunodiagnostic sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of soluble Taenia saginata antigens in stool samples (copro-antigens) of infected humans, using affinity-purified polyclonal antibodies obtained from rabbits hyperimmunized with excretory/secretory antigens derived from T. saginata maintained in vitro. Investigation of operating characteristics showed very low cross-reactivity with crude antigens from helminths other than Taenia, including Dipylidium caninum and Diphyllobothrium latum. The specificity of the assay was 95% when testing stool samples from 100 persons who were either infected with Ascaris lumbricoides, Trichuris trichiura, hookworms, Enterobius vermicularis or Hymenolepis nana, or who had no intestinal helminthosis detected. Analysis of diagnostic sensitivity demonstrated that in 85% of 34 samples from 23 untreated persons with intestinal T. saginata infection (selected by previous proglottid and/or egg detection) copro-antigens were detected by the T. saginata ELISA. In the same samples, Taenia eggs were detected in 62%. Only 41% of the samples reacted positively in a heterologous T. hydatigena ELISA. Post-treatment control revealed a high concentration of T. saginata copro-antigens for 1-4 d after administration of niclosamide or praziquantel, and negative values 9-17 d after treatment. The Taenia copro-antigens remained detectable by ELISA even after storage of untreated faeces at 25 degrees C for at least 5 d.