人抗乙脑病毒IgG抗体检测试剂盒的研制和应用

乙脑病毒SA14-14-2株疫苗原液经β丙内酯灭活Sepharose 4FF纯化后作为包被抗原,制备阳性替代品,应用间接ELISA法检测人血清中乙脑病毒抗体。建立内部质量控制血清标准,比较蚀斑减少中和试验(PRNT)与ELISA的相关性。检测46份乙脑相关血清的结果与国内同类试剂进行比较,阳性符合率为93.1%,阴性符合率为89.5%。在咸安地区3万多名2~14岁人群中进行乙脑病毒IgG抗体水平普查,阳性率22.5%,与国内同类试剂的符合率为95.7%,使用效果很好。     

中国新疆维吾尔自治区不明原因发热人群中Tahyna病毒感染状况调查

本研究通过对新疆地区不明原因发热人群血清Tahyna病毒抗体进行检测,以了解Tahyna病毒在当地的感染状况及分布特征。通过间接免疫荧光方法对新疆维吾尔自治区南部喀什地区、北部伊犁地区742例不明原因发热患者急性期血清标本进行Tahyna病毒抗体检测,并对IgM抗体阳性标本平行进行Tahyna病毒、Snowshoe hare病毒和Inkoo病毒三种抗原性相似的布尼亚病毒的蚀斑减少中和试验。研究结果显示新疆南部喀什地区采集不明原因发热患者急性期血清中,IgM抗体阳性率5.3%,IgG抗体阳性率18.3%;新疆北部伊犁地区采集的急性期患者血清标本中并未发现TAHV IgM抗体阳性;蚀斑减少中和试验结果显示,TAHV IgM抗体阳性患者血清中Tahyna病毒中和抗体滴度较其它两种布尼亚病毒中和抗体滴度升高明显。本研究证实新疆南部地区不明原因发热人群存在Tahyna病毒的急性感染和既往感染,为相关疾病的进一步监测提供基础。     

森林脑炎中和抗体蚀斑减少试验方法的建立及疫苗免疫人体后抗体检测

为评价森林脑炎疫苗的免疫效果,采用森林脑炎病毒的BHK细胞适应株,以BHK细胞为培养基质,建立了检测森林脑炎中和抗体的蚀斑减少试验方法,并用蚀斑减少法、小鼠法及ELISA法检测了原制森林脑炎灭活疫苗免疫人体后的中和抗体水平;免疫血清为按疫苗免疫程序免疫健康志愿者采血分离而制备。结果表明蚀斑减少法与小鼠法的特异性相近且敏感性较好、简便快速,缩短检测周期,为疫苗流行病学调查及新型疫苗的研究提供了快速的检测方法;原制灭活疫苗人体二针免疫后,人体血清中和抗体阳转率仅30％左右,急需提高现用原制灭活疫苗的质量。     

两种HSV-1及猴B病毒相关抗体测定方法的比较

目的以人单纯疱疹病毒（HSV-1）做为抗原,利用空斑法和IFA法比较猴BV和人HSV-1阳性血清两种不同血清的中和能力的差异,建立一种实用、准确、可靠的病毒毒力的检测方法。方法首先,将HSV-1病毒悬液作连续的10倍稀释,取1 mL接种于已经长成单层的Vero-E6细胞上,用1%甲基纤维素覆盖,待其出现蚀斑后计数,算出病毒悬液中每毫升所含蚀斑单位,即滴定出HSV-1的TC ID50。同时,用免疫荧光方法（IFA）对猴和人疱疹阳性血清进行滴定,得到其血清的效价。其次,用滴定出的病毒液分别与两种阳性血清体外中和后,接种到单层的Vero-E6细胞上,用1%甲基纤维素覆盖,待其出现蚀斑后计数。最后,计算出其蚀斑减少率。结果用1%甲基纤维素作覆盖层的蚀斑数量平均为10-5PFU,能形成115-116个/mL蚀斑,形状呈黍米大小的规则圆形,其蚀斑边缘清晰。IFA滴定的人HSV-1阳性血清与猴BV阳性血清的中和抗体均为1∶80。人HSV-1和猴BV两种阳性血清的空斑减少率均为100%。结论确定了利用1%甲基纤维素做为覆盖层可得到清晰可靠的蚀斑,由此方法检测到用人HSV-1可以代替猴B病毒,筛查猴B病毒抗体。且为将来进行药物筛选和中和实验中利用病毒空斑法建立方便、可靠的方法。     

流行性乙型脑炎组织培养中和试验方法研究

1．组织培养中和试验可以先将血清、病毒、维持液混合,然后一次加到细胞瓶中,简化操作,可节省人力、物力。 2．在血清稀释的中和试验中,可使用乙脑P,株鼠脑病毒,合适的病毒稀释度为10～,试验后第5—6日即可判定结果。 3．病毒稀释、血清稀释两种组织培养中和试验方法都可用于测定乙脑抗体,所得到的中和指数与中和效价,在强度上与小鼠脑内法所得到的中和指数是可比的。 4．血清稀释这个组织培养中和效价法,已证明适用于测知乙脑活疫苗免疫豚鼠、马匹、儿童血清中的乙脑中和抗体水平。     

检测流行性出血热病毒滴度和中和抗体效价的半微量空斑法

建立了检测流行性出血热病毒滴度和中和抗体效价的半微量空斑法。小牛血清与胎牛血清的培养效果无差异。7株不同来源的出血热病毒均能在E6细胞上形成空斑。接种的病毒浓度与形成的空斑数呈直线关系。用空斑法测得的病毒滴度稍低于荧光TCIE50滴定法。空斑减少中和试验的敏感性较荧光中和试验高30倍左右。同时还初步表明了本方法可用于流行性出血热病毒的抗原性分析。     

流行性出血热病毒的蚀斑形成技术

国外已有报道,流行性出血热病毒(EHFV)能在Vero-E6细胞上形成蚀斑。由于蚀斑试验和蚀斑减少试验用于测定病毒滴度及中和抗体效价较其它方法准确,且特异性高,适用于测定出血热病毒间抗原性差异,感染或免疫后中和抗体水平。但由于EHFV在细胞内繁殖培养时间较长,蚀斑形成的细胞培养条件要求较高,目前国内尚未见成功的报道。我们基本     

病毒中和抗体检测方法研究进展

疫苗接种在病毒性传染病防控中发挥了重要的作用,有效的疫苗在机体内能够快速诱导高效价的中和抗体,中和抗体水平是衡量疫苗免疫保护效果的关键指标。为应对疫苗的快速研发、大规模临床试验检测以及疫苗接种后人群免疫原性检测的需求,急需建立高灵敏度、高通量的病毒中和抗体评价方法。目前病毒中和抗体检测方法除传统的以活病毒为基础的噬斑抑制法和细胞病变法外,结合新技术发展,已经有很多新的方法,如含有报告基因的重组活病毒的检测方法和以假病毒为基础的中和抗体检测方法等,现对病毒中和抗体检测方法的研究作一综述。     

两种方法检测接种流行性出血热沙鼠肾细胞灭活疫苗后人体的中和抗体

本文应用空斑减少中和试验(PRNT)和细胞病变中和试验(cPENT)两种方法对出血热沙鼠肾细胞灭活疫苗扩大人体免疫后的血清进行中和抗体水平检测。根据两种方法对总计74人份的免疫后血清检测比较结果,两种方法检测的抗体阳转率和抗体水平(GMT)。CPENT法均高于PRNT法,经统计学处理均有显著性差异。不同免疫组的中和抗体水平比较结果,注射三针的阳转率(n=10,100％)高于两针组(n=10,20—30％);接种加氢氧化铝佐剂疫苗(n=13)较接种不加佐剂的两种疫苗(n=26)的抗体水平高,阳转率为92％—100％GMT为22—69;皮下途径(n=15)和肌肉途径(n=13)注射无明显差别,阳转率分别为87—93％和92—100％,GMT分别为29—46和22—61。以上结果进一步肯定沙鼠肾细胞疫苗的人体免疫性     

流行性乙型脑炎病毒血清抗体半微量空斑抑制法的改进

本文报道一种检测流行性乙型脑炎(简称乙脑)中和抗体的简化空斑抑制试验方法,用BHK21传代细胞在24孔塑料板上进行试验,在细胞未形成单层时,将病毒直接接种在刚消化的细胞悬液内,孵育后加甲基纤维素复盖物,再培养,细胞层经染色后即可计算空斑数。 用该法检查47名儿童乙脑疫苗免疫前后的中和抗体,与常规的鸡胚细胞全量法或BHK21传代细胞微量中和试验法作了比较,结果表明本法与其它常规法的敏感性一致,且具有简便快速、节省人力和原材料、空斑形成率高的优点,便于大量血清学检测。     

Evaluation of serological diagnostic test systems assessing the immune response to Japanese encephalitis vaccination

A new commercial anti-Japanese encephalitis virus IgM and IgG indirect immunofluorescence test (IIFT) was evaluated for the detection of the humoral immune response after Japanese encephalitis vaccination. The IgM IIFT was compared to two IgM capture ELISAs and the IgG IIFT was analysed in comparison to a plaque reduction neutralization test (PRNT50) and an IgG ELISA. Moreover, the course of the immune reaction after vaccination with an inactivated JEV vaccine was examined. For the present study 300 serum samples from different blood withdrawals from 100 persons vaccinated against Japanese encephalitis were used. For the IgM evaluation, altogether 78 PRNT50 positive samples taken 7 to 56 days after vaccination and 78 PRNT50 negative sera were analyzed with the Euroimmun anti-JEV IgM IIFT, the Panbio Japanese Encephalitis - Dengue IgM Combo ELISA and the InBios JE Detect IgM capture ELISA. For the IgG evaluation, 100 sera taken 56 days after vaccination and 100 corresponding sera taken before vaccination were tested in the PRNT50, the Euroimmun anti-JEV IgG IIFT, and the InBios JE Detect IgG ELISA. The Euroimmun IgM IIFT showed in comparison to the Panbio ELISA a specificity of 95% and a sensitivity of 86%. With respect to the InBios ELISA, the values were 100% and 83.9%, respectively. The analysis of the Euroimmun IgG IIFT performance and the PRNT50 results demonstrated a specificity of 100% and a sensitivity of 93.8%, whereas it was not possible to detect more than 6.6% of the PRNT50 positive sera as positive with the InBios JE Detect IgG ELISA. Thus, the IIFT is a valuable alternative to the established methods in detecting anti-JEV antibodies after vaccination in travellers and it might prove useful for the diagnosis of acutely infected persons.     

Neutralising antibodies for West Nile virus in horses from Brazilian Pantanal

Despite evidence of West Nile virus (WNV) activity in Colombia, Venezuela and Argentina, this virus has not been reported in most South American countries. In February 2009, we commenced an investigation for WNV in mosquitoes, horses and caimans from the Pantanal, Central-West Brazil. The sera of 168 horses and 30 caimans were initially tested using a flaviviruses-specific epitope-blocking enzyme-linked immunosorbent assay (blocking ELISA) for the detection of flavivirus-reactive antibodies. The seropositive samples were further tested using a plaque-reduction neutralisation test (PRNT90) for WNV and its most closely-related flaviviruses that circulate in Brazil to confirm the detection of specific virus-neutralising antibodies. Of the 93 (55.4%) blocking ELISA-seropositive horse serum samples, five (3%) were seropositive for WNV, nine (5.4%) were seropositive for St. Louis encephalitis virus, 18 (10.7%) were seropositive for Ilheus virus, three (1.8%) were seropositive for Cacipacore virus and none were seropositive for Rocio virus using PRNT90, with a criteria of ≥ four-fold antibody titre difference. All caimans were negative for flaviviruses-specific antibodies using the blocking ELISA. No virus genome was detected from caiman blood or mosquito samples. The present study is the first report of confirmed serological evidence of WNV activity in Brazil.     

Antibody to Viruses Affecting Cattle in Commercial Tissue Culture Grade Fetal Calf Serum

Commercial fetal calf serum (FCS) for tissue culture use was tested for neutralizing activity against several viruses which affect cattle. Certain lots of FCS contained no neutralizing activity, whereas other lots contained neutralizing activity to several viruses. It was concluded that the neutralizing activity found in certain lots of sera was due to specific antibody and that its presence could be most easily explained by the contamination of the FCS with serum from postcolostral bovine serum. A nonantibody inhibitor to vesicular stomatitis virus was also found at low levels in most lots of serum. Because those sera which had antibody had antibody to several viruses, it was suggested that the use of the micro-serum neutralization test with a few bovine viruses which are widespread in the bovine population should be satisfactory to detect FCS which was contaminated with postcolostral bovine serum.     

Adenosine phosphorylase activity in mycoplasma-free growth media for mammalian cells

Mammalian cells have enzymes that deaminate adenosine to inosine, which can readily be phosphorolysed to hypoxanthine. They do not, however, possess enzymes to form adenine by the cleavage of adenosine. For this reason, the release of adenine from adenosine by mammalian cell cultures has usually been interpreted as indicating the presence of mycoplasma, a frequent microbial contaminant that contains high levels of adenosine phosphorylase. We found that some human lymphoblast cultures free of mycoplasma showed high levels of adenosine cleavage and that this activity resulted from adenosine phosphorylase in the bovine serum used as the culture growth supplement. A survey of 13 serum supplements disclosed that fetal bovine serum (six lots) contains the highest adenosine phosphorylase activity, ranging from 9 to 648 nmol adenine produced per hour per ml serum; newborn calf serum (four lots) has much less activity, ranging from 0 to 5 nmol adenine produced per hour per ml serum; and donor horse serum (three lots) contains no detectable activity. These results suggest that mycoplasma tests dependent on the presence of adenosine phosphorylase or other enzyme activities may give false-positives with cultures containing fetal bovine serum supplements.     

Interferon γ in acute and subacute encephalitis

Intrathecal synthesis of interferon γ was shown in 14 out of 16 samples of cerebrospinal fluid collected in the first days of disease in adults, children, and newborn infants with herpes encephalitis. This synthesis was concomitant with that of interferon α and was switched off when the specific antibodies in the central nervous system increased. No endogenous interferon γ was detected in 11 serum samples or 13 samples of cerebrospinal fluid collected early in the course of the disease from patients with measles encephalitis and rubella encephalitis, or in serum and cerebrospinal fluid samples from seven patients with subacute sclerosing panencephalitis. In serum collected after the 10th day after the onset of neurological symptoms interferon γ was present at low concentrations in only three out of 11 serum specimens from patients with measles encephalitis or rubella encephalitis.Interferon γ was present in patients with acute herpes encephalitis and there was active virus replication, but it was not present in postinfectious encephalitis. Possibly the local production of specific antibodies masks the viral antigens and switches off the induction of interferons.     

Establishment of a national network of validated and qualified laboratories for neutralizing anti-vaccinia antibodies titration

A Proficiency Testing Study (PTS) was organized in France by the French Health Products Safety Agency (Afssaps) aiming at assessing the performance of laboratories in performing a neutralizing anti-vaccinia antibodies titration method by plaque reduction neutralization test (PRNT). The ultimate goal was to establish a national network of qualified and validated laboratories. Five laboratories were included in the PTS and four submitted their data. Three samples of human sera containing various immunoglobulin concentrations (a "high" serum: s-576, a "medium" serum: Ref-19584 and a "low" serum: s-483) were tested by PRNT as described in a procedure supplied by Afssaps and developed in each laboratory during preliminary assays. Data were sent to Afssaps which performed the statistical analysis. The overall performance of the four participating laboratories was satisfactory. This allowed the four participating laboratories to be validated and then to be qualified by the Ministry of Health. Finally a national network for anti-vaccinia immunoglobulins titration was established.     

A virus-type specific serological diagnosis of flavivirus infection using virus-like particles

Many flaviviruses are emerging and reemerging pathogens, such as West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members, plaque reduction neutralization test (PRNT) is the only available assay to determine the infecting flavivirus type.Since PRNT requires culturing raw viruses, it must be performed in biosafety level-3 or level-4 containment for many flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed flavivirus viral-like particles (VLPs) that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps: (ⅰ) VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h; (ⅱ)the neutralized VLPs are used to infect Vero cells; and (ⅲ) the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen (as quantified by the luciferase activities) is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the "gold standard" of the classic PRNT; importantly, it shortens the assay time from ＞10 days to ＜1 day, and can be performed in biosafety level-2 facility.     

Effect of genomic variation in the challenge virus on the neutralization titres of recipients of inactivated JE vaccines--report of a collaborative study on PRNT50 assays for Japanese encephalitis virus (JE) antibodies.

Japanese encephalitis (JE) viruses are grouped into four genotypes. Although currently available vaccines are derived only from viruses in genotype III, vaccines are known to protect against naturally occurring strains. Studies were undertaken to assess the suitability of a freeze-dried pool of human anti-JE plasma, collected from recipients of Biken (Nakayama-NIH) killed vaccine, to serve as an International Standard for antibodies to JE virus. Five participants in five countries submitted data from 11 assays on the candidate International Standard and seven coded samples including sera from recipients of vaccines containing a range of virus strains. The results of the study indicated that the 50% plaque reduction neutralization test (PRNT(50)titres) obtained for serum from recipients of killed vaccines, including the candidate standard, vary depending on the virus strain used in the neutralization tests, namely higher PRNT(50)titres were obtained when the challenge virus was homologous to the vaccine strain compared to use of a heterologous virus. Potencies expressed relative to the candidate standard are therefore affected by the strain of virus used in assays and the use of a standard would therefore not facilitate direct comparison of data from laboratories that have used different challenge strains.     

West Nile Virus Seroprevalence in the Greek Population in 2013: A Nationwide Cross-Sectional Survey

Cases of West Nile Virus (WNV) disease were recorded for three consecutive years in Greece following the year 2010 outbreak. A cross-sectional serologic survey was conducted to estimate the WNV seroprevalence and assess the ratio of infection to neuroinvasive disease. A stratified left-over sampling methodology was used including age and residence strata. A total of 3,962 serum samples was collected and tested for WNV Immunoglobulin G (IgG) antibodies by Enzyme–Linked Immunosorbent Assay (ELISA). All positive samples were further tested by Plaque Reduction Neutralization Test (PRNT) and WNV Immunoglobulin M (IgM) antibodies. WNV IgG antibodies were detected in 82 samples and 61 were also positive in PRNT representing a weighted seroprevalence of 2.1% (95% C.I.: 1.7–2.6) and 1.5% (95% C.I.: 1.2–2.0), respectively. Multivariable analysis showed that seroprevalence was associated with age and residence. The overall ratio of neuroinvasive disease to infected persons was estimated at 1:376 (95% C.I.: 1:421–1:338), while the elderly people had the highest ratio. This nationwide study provided valuable data regarding the epidemiology of WNV in Greece based on the fact that elderly people have higher risk of being both infected and having severe disease.     

Protective immunity of <Emphasis Type="Italic">E. coli</Emphasis>-synthesized NS1 protein of Japanese encephalitis virus

Immunogenicity and protective efficacy of recombinant Japanese encephalitis virus (JEV) NS1 proteins generated using DNA vaccines and recombinant viruses have been demonstrated to induce protection in mice against a challenge of JEV at a lethal dose. The West Nile virus NS1 region expressed in E. coli is recognized by these protective monoclonal antibodies and, in this study, we compare immunogenicity and protective immunity of the E. coli-synthesized NS1 protein with another protective immunogen, the envelope domain III (ED3). Pre-challenge, detectable titers of JEV-specific neutralizing antibody were detected in the immunized mice with E. coli-synthesized ED3 protein (PRNT50 = 1:28) and the attenuated JEV strain T1P1 (PRNT50 = 1:53), but neutralizing antibodies were undetectable in the immunized mice with E. coli-synthesized NS1 protein (PRNT50 < 1:10). However, the survival rate of the NS1-immunized mice against the JEV challenge was 87.5% (7/8), showing significantly higher levels of protection than the ED3-immunized mice, 62.5% (5/8) (P = 0.041). In addition, E. coli-synthesized NS1 protein induced a significant increase of anti-NS1 IgG1 antibodies, resulting in an ELISA titer of 100,1000 in the immunized sera before lethal JEV challenge. Surviving mice challenged with the virulent JEV strain Beijing-1 showed a ten-fold or greater rise in IgG1 and IgG2b titers of anti-NS1 antibodies, implying that the Th2 cell activation might be predominantly responsible for antibody responses and mice protection.