免疫酶斑点法检测汉坦病毒抗原的研究

目的 建立一种新的诊断肾综合征出血热(HFRS)的实验手段。方法 采用R22株、陈株和湖北株3种汉坦病毒接种家兔，制备兔抗汉坦病毒多克隆抗体(抗HTV-IgG)。然后采用混合的3种抗体进行免疫酶斑点法实验(IEDA)，检测患者血清和尿液中的汉坦病毒抗原。同时采用间接免疫荧光法(IFA)检测患者血清中抗汉坦病毒-IgM作对照。结果 经用相关分析，IEDA与IFA检测的结果高度相关。血中汉坦病毒抗原检出率为73．68％，尿中为65．00％。发病5d内，血中检出率为94．34％，尿中检出率为83．33％。5病日内的早期诊断率前者明显高于后者。结论 IEDA与IFA相比，阳性率比后者有较大提高，特别是5病日内的早期阳性率的提高明显，值得用于临床HFRS的早期诊断。     

肾综合征出血热IgM抗体早期诊断两步法MacELISA的建立

目的 建立和改善肾综合征出血热早期诊断IgM抗体捕获MacELISA法.方法 汉坦病毒核蛋白重组表达纯化后用辣根过氧化物酶标记,建立一种以酶标抗原为基础的MacELISA(称为两步法MacELISA)并与常规三步法MacELISA进行比较分析.结果 检测不同病日HFRS患者血清IgM抗体比较两步法与三步法MacELISA高度相关,敏感性和特异性为100%,无有明显差别.结论 本方法操作简单,用时少,成本低,适合用于肾综合征出血热早期诊断和汉坦病毒感染的监测.     

长春地区人群人类疱疹病毒6型抗体的检测

目的：建立检测血清中人类疱疹病毒6型（HHV-6）抗体的间接免疫荧光方法（IFA），检测人群中HHV-6抗体的水平。方法：用HHV-6GS株感染脐血单个核细胞制备抗原片，建立检测血清中HHV-6抗体的IFA法，并对长春市人群血清中的HHV-6抗体水平进行检测。结果：成功地建立了检测XHV-6抗体的间接免疫荧光方法，对长春市人群血清中的XHV-6抗体水平进行检测表明，XHV-6抗体阳性率为65．2％。结论：建立了特异性的IFA法，用于HHV-6感染的调查。     

抗汉坦病毒糖蛋白细胞内抗体的构建和稳定表达

目的　在内质网和细胞质内表达抗汉坦病毒糖蛋白G1,G2细胞内抗体。方法　PCR分别扩增抗汉坦病毒糖蛋白抗体VH和VL段基因 ,克隆入单链抗体表达载体pOPE 10 1 2 15 (Yol) ,转化大肠埃希菌XLI Blue ,IPTG诱导表达并鉴定其活性 ,再将单链抗体基因克隆入真核表达载体pEF myc ER和pEF myc CYTO ,转染VeroE6 ,使单链抗体在内质网和细胞质内得到表达 ;通过G4 18和有限稀释法筛选阳性克隆 ,建立稳定表达抗汉坦病毒糖蛋白细胞内抗体的细胞系。结果　Western blot检测证实原核表达单链抗体 ,ELISA ,IFA检测其具有抗原结合活性 ,真核表达显示内质网和细胞质内特异性荧光 ,筛选细胞系阳性率 >95 %。结论　成功建立稳定表达抗汉坦病毒糖蛋白细胞内抗体的细胞系 ,为利用汉坦病毒细胞内抗体研究抗病毒作用和病毒的包装复制及病毒感染机制奠定良好基础。     

重组汉坦病毒核衣壳蛋白抗原用于胶体金标记免疫层析法检测肾综合征出血热抗体

目的制备高表达量、高活性的HV NP重组抗原,以此为基础建立一个可同时检测HFRs患者血清中IgM和lgG抗体的快速胶体金标记诊断试剂盒。方法从TA-A537中扩增出截短的HV S基因片段P6-119,定向克隆至原核表达载体pET-30a中进行原核表达,采用亲和层析法纯化重组蛋白。以胶体金标记重组抗原,应用金标快速免疫层析法同时检测HFRS患者血清中IgM和IgG抗体。结果金标快速免疫层析法可同时检测HFRS患者血清中IgM和IgG抗体,与IFA比较,IgG抗体检测的敏感性和特异性分别为94.9%、100%。与ELISA比较,IgM抗体检测的敏感性和特异性均为100%。结论截短的重组汉坦病毒NP蛋白表达量高且具有良好的抗原性。以此为基础建立的金标快速免疫层析法显示出很高的敏感性和特异性,且具有简便、快速等优点,非常适用于各种层次尤其是缺乏实验条件和专业人员的基层医疗单位对HFRS疑似患者作出早期诊断。     

微斑免疫酶技术在检测肾综合征出血热病毒及其特异抗体中的应用

本文报道一种简便且快速的方法—微斑免疫酶技术(MPIPA),检测Vero-E6细胞培养的肾综合征出血热(HFRS)病毒及其特异抗体,并与免疫荧光技术(IFA),酶联免疫吸附技术(ELISA)进行比较。结果,本法用于检测HFRS患者血清IgG抗体和抗HFRS病毒单克隆抗体(McAb),其敏感性介于ELISA和IFA之间;用于检测Vero—E6细胞培养的病毒,阳性结果比IFA和细胞病变(CPE)出现早。MPIPA具有简单,快速、敏感性高、特异性好,结果易判定诸优点,便于基层医疗单位推广应用,适合于临床HFRS血清标本的检测和血清流行病学调查。     

酶联捕获法检测抗人巨细胞病毒-IgM抗体的实验分析

目的 建立检测人巨细胞病毒(HCMV)抗原特异性IgM抗体的酶联捕获技术。方法 应用酶联捕获法检测68例巨细胞病毒感染者血清中抗HCMV-IgM，并与常用的间接ELISA法进行比较。结果 酶联捕获法特异性及敏感性均高于间接ELISA法，且不受RF因子的影响。结论 酶联捕获法敏感性高、特异性强、简便、快速，重复性好，具有实际应用价值。     

基于荧光微球的病毒性出血热IgM抗体检测方法的建立

目的 建立并初步评价多元检测引起病毒性出血热病原体特异性IgM抗体的方法.方法 在原核细胞中重组表达纯化马尔堡病毒、拉沙热病毒、裂谷热病毒、肺综合征出血热汉坦病毒、汉滩病毒、Seoul病毒及普马拉病毒核蛋白(NP),共价偶联到7种不同的xMAP荧光微球上.优化评价偶联效果,通过参比血清中相应病毒NP特异性抗体检测,评估检测方法,并与常规使用的MacELISA方法进行比较.结果 在Luminex平台的基础上建立了多元检测引起病毒性出血热的病毒核蛋白特异性抗体的方法,特异性与敏感性与常规应用的特异性抗体检测MacELISA试剂盒相当,但可以同时排查多个病毒感染情况.结论 利用Luminex xMAP技术建立基于病毒核蛋白多元检测方法快速敏感,操作简单,可以用于病毒性出血热的检测和血清流行病学调查.     

化学发光法检测抗-HEV IgG方法的建立

目的 建立化学发光检测抗HEV IgG方法,用于HEV感染的实验室诊断及流行病学调查,为试剂盒研制打下基础.方法 以HEV重组抗原包被微孑孔板,辣根过氧化物酶标记的单克隆抗人IgG抗体为第二抗体,建立抗HEV IgG的化学发光检测方法,评价其灵敏度、特异性、精密性等指标.检测患者血清中抗HEV IgG抗体并与第三方试剂比较.结果 建立了检测抗HEV IgG的化学发光方法,检测500例临床患者标本并与对照试剂盒对比,阳性符合率为99.32％,阴性符合率为98.58％,总符合率98.80％,其灵敏度、特异性、重复性均达到设计要求.结论 建立了化学发光检测血清中抗-HEV IgG的方法,具有敏感性高、特异性强、操作简便等特点,适用于戊型肝炎的临床诊断和流行病学调查.     

以汉坦病毒重组核衣壳蛋白为抗原进行ELISA血清分型的研究

目的 以汉坦病毒重组核衣壳蛋白（NP）为抗原，对陕西、河北两省部分地区HFRS患者的血清进行ELISA分型。方法 用5种汉坦病毒NP为抗原，建立一种间接ELISA法，并检测上述地区患者血清中特异性IgC。结果陕西、河北两省部分地区汉坦病毒的感染以血清型HTN为主，其次为SEO型，并检测出2例疑似DOB型感染的血清。结论 用汉坦病毒NP作抗原进行ELISA血清分型是可行的，并首次发现我国存在DOB血清型的免疫学证据。     

Comparison of human sera reactivities in immunoblots with recombinant human herpesvirus (HHV)-8 proteins associated with the latent (ORF73) and lytic (ORFs 65, K8.1A, and K8.1B) replicative cycles and in immunofluorescence assays with HHV-8-infected BCBL-1 cells

The development of reliable, sensitive, and specific serological methods for the detection of human herpesvirus-8 (HHV-8) antibodies is critical for a thorough understanding of HHV-8 prevalence and pathogenesis. To evaluate the potential usefulness of HHV-8 proteins in measuring the responses against both latent and lytic antigens, we selected 1 latent [open reading frame (ORF) 73] antigen and 3 HHV-8 lytic antigens (ORFs 65, K8.1A, and K8.1B) previously identified as immunogenic [Virology (1998) 243, 208-217]. Full-length genomic ORF 73 and full-length ORFs 65, K8.1A, and K8.1B from the cDNA clones were cloned, expressed in bacterial and baculovirus-insect cell expression systems, and purified as GST fusion proteins. These recombinant proteins were used in Western blot reactions to test sera from 104 human immunodeficiency virus (HIV)+/Kaposi's sarcoma (KS)+ homosexual men, 77 HIV+/KS- homosexual men, and 84 age-matched HIV-/KS- men. These sera were also tested in immunofluorescence assays (IFAs) with uninduced and 12-O-tetradecanoylphorbol-13-acetate-induced B cell lymphoma-1 cells to detect antibodies against latency-associated nuclear antigens (LANA) and antibodies against lytic antigens (cytoplasmic fluorescence). These sera exhibited differential reactivities reflecting different titers of antibodies against HHV-8 proteins, and variable reactivities were seen more commonly with the sera from HIV-/KS- adult men. In the Western blot assay, 89% (93 of 104) of HIV+/KS + sera, 60% (46 of 77) of HIV+/KS- sera, and 7% (6 of 84) HIV+/KS- sera were reactive with both latent and lytic recombinant antigens. Western blot reactions with ORF 73 protein were more sensitive than LANA-IFA results. The lytic IFA and lytic Western blot (ORFs 65 and K8.1A) assays were more sensitive than the ORF 73 Western blots and LANA-IFA. With an exception of 2 sera from the HIV-/KS- group, all sera positive for lytic IFA antibodies and ORF 65 and K8.1A antibodies were also positive for latent antibodies. With few exceptions, sera positive for ORF 65 antibodies were also positive for K8.1A antibodies, and sera recognized the K8.1A protein more often than the K8.1B protein. There is a high degree of concordance between IFA and Western blot reactions, suggesting that this panel of HHV-8 recombinant proteins could detect a majority of the HHV-8-seropositive individuals. These results suggest that IFA followed by confirmation with the Western blot reactions with a panel of latent and lytic immunogenic antigens would provide a reliable, sensitive, and specific method for the detection of HHV-8 antibodies.     

A comparison of the IFA and the ELISA for the demonstration of antibodies against schistosome gut-associated polysaccharide antigens in schistosomiasis

The applicability of two immunodiagnostic techniques was studied for the detection of antibodies against schistosome gut-associated polysaccharide antigens in human schistosomiasis mansoni: the immunofluorescent antibody reaction (IFA) using Rossman's fixed paraffin sections of adult worms and the enzyme-linked immunosorbent assay (ELISA) with a trichloroacetic acid soluble fraction of total adult worm antigens (AWA-TCA).With the IFA, gut-associated polysaccharide antigens could be demonstrated with an anti-IgM conjugate in a high percentage of the sera tested, although false-negative reactions were occasionally recorded. The use of an anti-IgG conjugate resulted in the demonstration of antibodies against additional antigens in the parenchyma of the worm and on the tegument. Specific IgM antibodies were present in higher concentrations in the sera from children than in those from adults.Using AWA-TCA as the antigen preparation in the ELISA, only antibodies against the circulating anodic antigen (CAA) could be demonstrated. Pretreatment of the ELISA-plates with poly-L-lysine to couple AWA-TCA was not necessary. The ELISA was sucessfully applied with anti-Ig, anti-IgG and anti-IgM conjugates. With anti-Ig conjugate the test was very sensitive and gave less false-negative reactions than the IFA. There was a significant difference between Ig, IgG, and IgM titres of children and adults. The use of an immunogalactosidase assay with a fluorogenic substrate in the ELISA, resulted in a test which was able to detect antibodies at ten times higher dilutions than with the immunoperoxidase assay.This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases     

Hantavirus infections in Spain: analysis of sera from the general population and from patients with pneumonia, renal disease and hepatitis.

BACKGROUND: Hantaviruses are rodent borne viruses in the family Bunyaviridae that cause significant morbidity in large areas of Europe. There are only a few reports available on hantavirus infections from Spain. Although the results of these earlier studies indicated the presence of hantavirus infections, no confirmative or serotype-specific analyses have been performed. OBJECTIVES: To investigate whether hantaviruses cause human infection/disease in Spain. STUDY DESIGN: Ten thousand, four hundred and eighteen serum samples from the general population and 599 sera from 492 patients with potential hantavirus infections (renal disease, pneumonia or hepatitis) were initially screened by immunofluorescence assay (IFA) using Hantaan, Seoul and Puumala hantavirus antigens. Altogether 193 suspicious samples (165 from healthy people and 28 from patients) were selected for confirmation by quality-assured assays. RESULTS AND CONCLUSIONS: Of the 165 pre-screened serum samples from healthy individuals, only five could be confirmed by IFA for hantavirus-reactive antibodies (using Dobrava, Saaremaa, Hantaan or Puumala virus antigens). In addition, one serum was found weakly positive for hantavirus-reactive IgG by ELISA using recombinant Saaremaa virus (SAAV) nucleocapsid (N) antigen, and subsequently confirmed by immunoblotting. Thus, the results indicated a low (0.06%) total antibody prevalence to hantaviruses in Spain. Of 28 pre-screened serum samples from hospitalized patients, eight reacted as positive or showed border-line reactivities for hantavirus-specific IgM by ELISA using recombinant Saaremaa and Puumala virus N antigens. The IFA/ELISA reactive/border-line samples were subsequently analyzed by a focus reduction neutralization test, which revealed low titers (1:80) against SAAV in two samples from a patient with hepatic disease. The nature of the hantavirus(es) potentially involved remain, however, unknown, since none of the positive samples showed neutralizing titers of the expected range to any of the known European hantaviruses.     

Novel serological tools for detection of Thottapalayam virus, a Soricomorpha-borne hantavirus

We developed serological tools for the detection of hantavirus-specific antibodies and hantavirus antigens in shrews. The work was focussed to generate Thottapalayam virus (TPMV)-specific monoclonal antibodies (mAbs) and anti-shrew immunoglobulin G (IgG) antibodies. The mAbs against TPMV nucleocapsid (N) protein were produced after immunization of BALB/c mice with recombinant TPMV N proteins expressed in Escherichia coli, baculovirus and Saccharomyces cerevisiae-mediated expression systems. In total, six TPMV N-protein-specific mAbs were generated that showed a characteristic fluorescent pattern in indirect immunofluorescence assay (IFA) using TPMV-infected Vero cells. Out of the six mAbs tested, five showed no cross-reaction to rodent-associated hantaviruses (Hantaan, Seoul, Puumala, Tula, Dobrava-Belgrade and Sin Nombre viruses) in IFA and enzyme-linked immunosorbent assay (ELISA), although one mAb reacted to Sin Nombre virus in IFA. None of the mAbs cross-reacted with an amino-terminal segment of the shrew-borne Asama virus N protein. Anti-shrew-IgG sera were prepared after immunization of rabbits and BALB/c-mice with protein-G-purified shrew IgG. TPMV-N-protein-specific sera were raised by immunisation of Asian house shrews (Suncus murinus) with purified yeast-expressed TPMV N protein. Using these tools, an indirect ELISA was developed to detect TPMV-N-protein-specific antibodies in the sera of shrews. Using an established serological assay, high TPMV N protein specific antibody titres were measured in the sera of TPMV-N-protein-immunized and experimentally TPMV-infected shrews, whereas no cross-reactivity to other hantavirus N proteins was found. Therefore, the generated mAbs and the established ELISA system represent useful serological tools to detect TPMV, TPMV-related virus antigens or hantavirus-specific antibodies in hantavirus-infected shrews.     

Kaposi's sarcoma-associated herpesvirus serology in Europe and Uganda: multicentre study with multiple and novel assays

A multicentre study was undertaken to define novel assays with increased inter-assay concordance, sensitivity, specificity and predictive value for serological diagnosis of human herpesvirus type 8 (HHV-8) infection. A total of 562 sera from European and Ugandan human immunodeficiency virus (HIV)-infected or uninfected individuals with or without Kaposi's sarcoma (KS) and blood donors were examined under code by 18 different assays in seven European laboratories. Sera from KS patients and all non-KS sera found positive by at least 70%, 80%, or 90% of the assays were considered "true positive." The validity of the assays was then evaluated by univariate logistic regression analysis. Two immunofluorescence assays (IFA) for detection of antibodies against HHV-8 lytic (Rlyt) or latent (LLANA) antigens and two enzyme-linked-immunosorbent assays (ELISA) (M2, EK8.1) for detection of antibodies against HHV-8 structural proteins were found to be highly concordant, specific, and sensitive, with odds ratios that indicated a high predictive value. When used together, the two IFA (Rlyt-LLANA) showed the best combination of sensitivity (89.1%) and specificity (94.9%). The performance of these assays indicate that they may be used for the clinical management of individuals at risk of developing HHV-8 associated tumours such as allograft recipients.     

Evaluation of two commercially available immunoassays for the detection of hantavirus antibodies in serum samples.

BACKGROUND: hantaviruses are members of the family Bunyaviridae and the spectrum of clinical symptoms in humans may vary from sub-clinical to severe haemorrhagic fever with renal syndrome (HFRS) or pulmonary syndrome (HPS). Several serotypes have been described from which at least five are pathogenic to humans. Each serotype has a different animal reservoir and geographical distribution. In the acute phase of the disease the clinical diagnosis may be confirmed by serology or by polymerase-chain reaction (PCR). OBJECTIVE: to evaluate two commercially available immunoassays using sera from hantavirus suspected and non-hantavirus patients: an enzyme immunoassay (EIA) developed by MRL Diagnostics, for the detection of immunoglobulins M (IgM) and G (IgG) against several hantavirus serotypes and an indirect immunofluorescence assay (IFA) from Progen, based on slides coated with Hantaan virus (HNTV) and Puumala virus (PUUV), infected cells. STUDY DESIGN: a total of 145 serum samples were used for this study. The serum panel included serum samples from patients suspected of mild (n=91), severe (n=10) HFRS and patients with other viral infections (n=44). RESULTS: the agreement between the MRL EIA and the Progen IFA for the detection of IgM and IgG serum antibodies ranged from 87 to 91%, respectively. In the non-hantavirus group one out of 44 samples was positive by the Progen HNTV IgM IFA, none in the Progen PUUV IFA and two samples in the MRL IgM EIA, resulting in specificities of 98, 100 and 95%, respectively. The sensitivities and specificities of the MRL EIAs compared to the Progen overall PUUV and HNTV IFAs were 90 and 91% for IgM, respectively, and 96% for IgG in both immunoassays. CONCLUSIONS: the MRL EIA proved to be relatively sensitive and specific assay for the serological diagnosis of mild and severe HFRS.     

New immunofluorescent antibody test for diagnosis of candidiasis using macrophage-engulfed antigens as substrate

A new method for detection of antibodies to polysaccharidic antigens of Candida albicans is described. The method is based on the selective uptake of carbohydrate-rich antigens by mouse peritoneal macrophages, the antibodies being revealed by indirect immunofluorescence after incubation of fixed, antigen-containing macrophages with sera (MU/IFA test). This test shows greater sensitivity than counterimmunoelectrophoresis (CIE) with good discrimination between infected patients and normal asymptomatic individuals. The combination of CIE using cytoplasmic antigens and MU/IFA using surface antigens is useful in a considerable majority of cases for diagnosis of clinical infection.     

Enzyme immuno assay for the detection of virus specific IgG and IgM antibody in patients with Haemorrhagic Fever with Renal Syndrome

Summary Consecutive serum samples collected from 235 patients with Haemorrhagic Fever with Renal Syndrome (HFRS), between two days and two years after onset of disease, have been analysed for the presence of IgG and IgM type of antibodies specific for Hanta-viruses. The sera were screened in parallel by a newly developed indirect Immuno Enzyme Assay (EIA) in parallel with Indirect Immunofluorescent Antibody Assay (IFA). In both tests the Hantaan virus strain 76–118 was used as the antigen. The EIA was much more sensitive than the IFA test for the detection of IgM type antibodies. With the indirect EIA IgM type antibodies against Hantaan virus 76–118 have been detected in HFRS patient's sera from the second day of illness indicating the usefulness of this test for the early serological diagnosis of this disease.