快速荧光灶抑制试验的应用和改进

快速荧光灶抑制试验(rapid fluorescent focus inhibition test,RFFIT)是世界卫生组织(World Health Organisation,WHO)推荐的狂犬病病毒中和抗体检测标准方法,已被应用于狂犬病疫苗免疫效果评估、狂犬病疫苗新型免疫方案评估、狂犬病诊断试剂和方法评估、狂犬病被动免疫制剂评价.对RFFIT的改进包括自动化判读结果和用表达绿色荧光蛋白(green fluorescent protein,GFP)的重组狂犬病病毒做为攻击病毒,这些改进目前尚未获得广泛应用,传统RFFIT仍然是值得推广的狂犬病病毒中和抗体检测方法.     

ELISA法与RFFIT法检测狂犬病疫苗免疫后血清抗体的比较

目的比较狂犬病疫苗免疫后血清抗体酶联免疫吸附试验（ELISA）与快速免疫荧光灶抑制试验（RFFIT）两种检测方法。方法对93名研究对象采用0、3、7、14和28d程序进行暴露后免疫，分别采用ELISA法与WHO认可的金标法RFFIT法检测免疫DO、D3、D7、D14、D45d的血清抗体。结果免疫DO、D3、D7、D14、D45dELISA法检测抗体阳性率分别为8．6％、11．8％、22．6％、49．6％、86％，RFFIT法检测抗体阳性率分别为0％、0％、22．58％、100％、100％，ELISA法与RFFIT法阳性符合率分别为0％、0％、34.8％、100％、100％；阴性符合率分别为100％、100％、81．4％、0％、0％。结论ELISA法检测狂犬病疫苗免疫后血清抗体，免疫早期DO、D3、D7假阳性率较高，免疫D14、D45则假阴性率较高。建议采用WHO认可的方法做检测。     

利用RV-IgG标准品对风疹疫苗免疫后血清血凝抑制抗体保护浓度的测定

目的 利用风疹病毒的IgG标准品(RV-IgG)进行定量ELISA试验和血凝抑制试验,以测定风疹疫苗免疫后血清血凝抑制(HI)抗体的保护浓度,并验证试剂盒的准确性.方法 RV-IgG标准品和10对风疹疫苗免疫前后血清的定量ELISA试验;风疹病毒血凝素的粗提以及血凝试验;RV-IgG标准品的血凝抑制试验测定其血凝完全被抑制时RV-IgG浓度.结果 将RV-IgG标准品稀释为8个不同浓度梯度做定量ELISA,所测得的浓度值与已知浓度相比无统计学意义(P ＞0.05);10对风疹疫苗免疫前后人血清抗体浓度进行比较,免疫后血清抗体浓度显著升高(t =2.151,P＜0.05);风疹病毒血凝素的凝集效价为1∶128,RV-IgG标准品血凝抑制效价为1∶32,对应的HI抗体浓度为25 IU/ml.结论 实验结果证实血清中风疹病毒HI抗体保护浓度为25 IU/ml;风疹疫苗初次免疫后血清抗体保护性阳转率可达到80％.     

乙型肝炎病毒表面抗原国家定量标准品的研制

目的 研制乙型肝炎病毒表面抗原(HBsAg)国家定量标准品及定量线性参考品.方法 收集各地乙型肝炎患者和健康献血员血样,用不同厂家乙型肝炎、丙型肝炎病毒血清学试剂盒筛选.6个协作单位用7种试剂以世界卫生组织(WHO)HBsAg定量标准品标定1份HBsAg国家定量标准品,稀释后得到HBsAg国家定量线性参考品.结果经7种试剂共21次协作标定,得到HBsAg国家定量标准品的浓度为1226 IU/ml,各次检测结果的变异系数均小于15%.将国家定量标准品系列稀释后得到由8个不同浓度血清组成的HBsAg国家线性参考品,经检测后表明其浓度与理论浓度的差值均<15%,通过加速试验检测该参考品的稳定性效期暂定为5年.将其作为HBsAg国家线性参考品并初步限定检测结果的允许值范围.结论 标定1份HBsAg国家定量标准品并建立了由8份血清组成的HBsAg国家定量线性参考品.     

新型冠状病毒抗体检测结果的标准化和平行比较北大核心CSCD

目的通过世界卫生组织(world health organization,WHO)新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)抗体国际标准品(international standard,IS)(G样本)、抗体参考盘(样本E、F、H、I、J)的应用降低SARS-CoV-2抗体检测结果差异、提高检测结果一致性。方法利用基于SARS-CoV-2活病毒的微量中和试验、假病毒中和试验以及商品化的ELISA试剂盒对WHO的SARS-CoV-2抗体IS和参考盘等共10个样本(A~J)分别进行检测,对检测结果进行分析比较。结果以IS为参考品确定其他样本的相对浓度,降低了检测结果之间的差异。假病毒中和试验、ELISA检测血清抗体参考盘结果与基于SARS-CoV-2 HB02株的中和试验基本一致,个别试剂可检测弱阳性样本。结论SARS-CoV-2抗体IS的使用促进了血清学检测方法的标准化,抗体参考盘适用于基于SARS-CoV-2原型株的所有检测方法,假病毒中和试验、ELISA等在一定程度上可作为活病毒中和试验的替代方法。     

肾综合征出血热Ⅰ型灭活疫苗免疫后4年抗体水平监测

目的 比较肾综合征出血热（HFRS）Ⅰ型灭活疫苗接种组和对照组免后4年抗体水平,了解HFRSⅠ型灭活疫苗有无增强接种组隐性感染发生情况。方法 1994年7月-1998年7月在建德市肾综合征出血热Ⅰ型灭活疫苗随机对照试验现场,采集接种组和对照组双份血清各305和283人。分别按对照组免前间接荧光抗体阳性和阴性的第二份血清间接荧光抗体滴度分布情况确定阳性判断阈值（cut-off值）,由此判断免前间接荧光抗体阳性和阴性的接种组人群发生隐性感染情况。结果 以不同的cut-off值来比较接种组和对照组免前间接荧光抗体阳性人群的抗体阳性率,差异无显著性,在免前间接荧光抗体阴性接种组和对照组中,其差异也无显著性,未见接种组和对照组免后抗体几何平均滴度和抗体阴转率差异有显著性。结论 接种组同对照组免后4年荧光抗体水平差异无显著性,未发现HFRSⅠ型灭活疫苗增强接种人群隐性感染发生率。     

RANA和EBNA抗体检测在类风关等疾病中的应用

<正>类风湿性关节炎相关核抗原（RANA）是一种受EB病毒诱导存在于淋巴细胞内的核抗原。许多研究证明类风湿性关节炎（RA）患者血清中有高滴度RANA抗体。RANA与EB病毒抗原特别是EB病毒核抗原（EBNA）有密切关系。Catalano等报告,RANA抗体与EBNA抗体滴度变化有关。本文用免疫荧光技术（IIF）检测RA及其他疾病中RANA抗体和EBNA抗体,并探讨其临床意义。1材料和方法1.1对象 RA患者68例,男12例,女56例。其他结缔组织病70例,包括干燥综合征（SS）7例;系统性红斑狼疮（SLE）24例;强直性脊椎炎（AS）20例;骨关节炎（OA）15例;硬皮病（PSS）4例。鼻咽癌（NPC）18例。正常人 51例。收集血清-30℃保存待测。1.2 RANA抗体检和使用IIF法。Raji细胞涂片。RANA抗体阳性可见大多数细胞核及胞浆内出现分布均匀的密集的细小颗粒型荧光。阳性参考血清由301医院栗占国赠送。1.3 EBNA抗体的检测 参照 Catalano方法,应用抗补体间接免疫荧光法。EBNA抗体阳性可见大多数细胞核出现均匀的荧光。1.4 阳性标准RANA 抗体和EBNA抗体均以≥1:10为阳性。阳性血清滴度取对数,并计算平均滴度。1.5统计学方法 所有资料均经方差分析或多元逐步回归,计算相关性及显著性。2结果2.1各组疾病及正常人RANA抗体和EBNA抗体阳性率结果见表1。     

不同基因型戊型肝炎病毒重组抗原p166用于抗体检测的价值

目的探讨不同基因型和亚型戊型肝炎病毒（HEV）ORF2重组蛋白p166用于抗体检测的价值,为研发准确可靠的戊型肝炎诊断试剂提供新的途径.方法用等浓度的不同基因型和亚型HEV p166作为包被抗原,对血清标本进行酶联免疫吸附试验测定.用HEV多基因型通用性引物逆转录套式PCR（RT-nPCR）扩增标本中HEV RNA,并测序、分型.结果8种不同p166抗原对30份健康献血者血清无抗原性,对182份来自世界不同国家和地区的已知HEV抗体阳性血清和7份HEV实验感染动物血清标本均呈阳性反应,但所得血清抗体滴度的高低与所用抗原的基因型有明显关系.RT-nPCR检测的50份中国血清标本中,19份阳性,基因分型均为Ⅳ型,与Ⅳ型中国株p166抗原反应最好.而以同属于第Ⅲ基因型的猪HEV新西兰株和人HEV美国株重组p166检测血清标本,两者结果差异无统计学意义.以多基因型p166混合抗原建立的ELISA抗体检测法与两种市售试剂盒比较,前者敏感性高,特异性好.结论不同基因型和亚型的HEV重组蛋白p166对不同血清标本HEV抗体检测的敏感性高低不同,因此多基因型和亚型p166的混合抗原是HEV抗体检测的最佳抗原.     

人类免疫缺陷病毒1型RNA定量参考品的研制

目的　研制人类免疫缺陷病毒 1型 (HIV 1)RNA核酸国家参考品及制定相应标准。方法　收集各地HIV感染者阳性血浆和HIV非感染者血浆 ,应用HIV、HCV抗体和HBsAg检测试剂进行筛选 ,对HIV抗体筛查阳性者用新加坡Genelabs公司的HIVBLOT 2 2确证试剂进行确证。以世界卫生组织 (WHO)推荐的HIVRNA标准品对国家HIV核酸参考品中定量样品进行标定 ,并对其稳定性进行研究。结果　经过筛选 ,选出 8份样品为阴性参考品 ,8份样品为阳性参考品 ,3份为定量参考品 ,6份为灵敏度参考品 ,5份为线性参考品。几次独立标定 ,得到定量参考品HIVRNA的国际单位(IU) ,其中b1～b3的国际单位的对数值在 x±s以内 ,表明结果可靠。稳定性实验数据表明 ,该核酸参考品在 4℃以下可存放 4d。结论　初步建立了HIV核酸参考品 ,这将对HIV核酸诊断试剂的质量评价提供重要依据     

脊髓灰质炎病毒抗体免疫荧光检测法的特性及其应用的研究

用自制脊灰病毒抗原片，通过免疫荧光技术检测ＩｇＧ或ＩｇＭ类脊灰抗体。并与中和试验对比，在受检的自然人群和服苗儿童１８８份血清标本中，两法检出脊灰Ⅰ－Ⅲ型抗体ＩｇＧ的阳性率无显著性差异，总符合率为９４～９６％，测得的抗体滴度呈高度正相关。用本法检测８份疑似脊灰早期病例血清中脊灰抗体ＩｇＭ，与用ＥＬＩＳＡ测定的结果一致。提示免疫荧光法可用於脊灰抗体水平测定和病例的血清学诊断。     

A rabies-specific human monoclonal antibody that protects mice against lethal rabies.

According to a recommendation from WHO (World Health Organisation) for prevention of a possible rabies infection, active vaccination has to be combined with application of immunoglobulin to get a fast protective effect. At present, preparations of purified human or equine rabies-specific immunoglobulin are used. We have generated a human rabies-specific monoclonal antibody (huMAb) by immortalization of human B-cells with Epstein Barr Virus (EBV), followed by fusion with a mouse myeloma cell. The resulting clone TW-1 secrets an IgG1 lambda huMAb which specifically reacts in ELISA with 5 laboratory rabies virus strains of serotype 1 and DUV3 (Duvenhage, serotype 4). Western Blot analysis revealed fine specificity for the G glycoprotein (gp67) of rabies virus. HuMAb TW-1 neutralizes rabies virus in vitro (RFFIT) as well as in vivo and protects rabies infected mice. Compared to polyclonal human rabies immunoglobulins, huMAb TW-1 is advantageous, because of its defined specificity and the very low amounts of total protein needed for therapeutic effects.     

Rapid diagnosis of rabies and post-vaccinal encephalitides.

In an attempt to establish the diagnoses of rabies post-vaccinal encephalitis (PVE) and early rabies encephalitis, paired serum and CSF levels of rabies neutralizing antibody (Rab) and rabies specific-IgM (RIgM) were compared in 12 PVE, 10 rabies and five control patients with similar presenting clinical features. Rapid methods of rabies antigen detection were evaluated in 17 patients. All 12 PVE patients had Rab in their serum and in eight it was also present in the CSF. These same eight had RIgM in the serum, and in seven also in the CSF. The CSF antibodies may have originated in the plasma since six patients had a high albumin quotient indicating leakage across the blood-brain barrier. Among the rabies patients, only the two vaccinated ones had serum Rab; this was also detected in the CSF of one and RIgM was in the CSF of the other. A raised IgG Index, indicating intrathecal synthesis of IgG was seen in five of 12 PVE patients. This did not correlate with the presence of CSF rabies antibody, suggesting production of antibody to other vaccine antigens of neural origin. The diagnosis of rabies encephalitis in life was made by antigen detection in a skin biopsy. No false positive results occurred and the method was as efficient as immunofluorescence of a post-mortem brain biopsy.     

Comparative evaluation of a simple indirect immunofluorescence test and mouse neutralization test for assaying rabies antibodies

In this study, we have developed and evaluated a simple indirect immunofluorescence test (IIFT) to detect rabies antibodies in a two-step immunofluorescence assay. One hundred and eighty five serum samples from people who had taken different rabies vaccines and 8 pairs of serum and CSF samples from confirmed paralytic rabies cases were tested by IIFT and results evaluated in comparison to standard mouse neutralization test (MNT). Though the titres of rabies antibodies obtained with IIFT were 2-4 times lesser in comparison to MNT, a significant correlation was seen between the two tests (R = 0.883). The specificity of this IIFT was found to be 97.9% and the sensitivity was 97.2%. These results indicate that this simple and rapid IIFT can be used to screen large number of serum samples to monitor sero-conversion after pre or post exposure vaccination and may also assist in rapid ante-mortem diagnosis of atypical human rabies.     

狂犬病高发地区犬只感染情况调查分析

目的 分析自然状况下犬只感染狂犬病毒状况及其与人间狂犬病的关系.方法 2005年10月到2006年12月间,在狂犬病疫情最严重的贵州、广西和湖南3省区的狂犬病高、中、低发病地区分别选择1-2个市作为标本采样点,用直接免疫荧光法和RT-PCR两种方法 检测标本.结果 在3省区的15个市共收集犬脑标本2887份,检测阳性标本66份,感染率为2.3%,不同地区犬感染率与当地人狂犬病发病率不呈线性相关.结论 犬只感染狂犬病毒普遍存在,这是我国狂犬病高发区疫情严重的一个重要因素.     

Alum adjuvanted rabies DNA vaccine confers 80% protection against lethal 50 LD50 rabies challenge virus standard strain

Rabies is a serious concern world-wide. Despite availability of rabies vaccines for long; their efficacy, safety, availability and cost effectiveness has been a tremendous issue. This calls for improvement of rabies vaccination strategies. DNA vaccination has immense potential in this regard. The DNA vaccine pgp.LAMP-1 conferred 60% protection to BALB/c mice against 20 LD₅₀ rabies challenge virus standard (CVS) strain challenge. Upon supplementation with Emulsigen-D, the vaccine formulation conferred complete protection against lethal challenge. To assess the feasibility of this vaccine formulation for human use, it was tested along with other FDA approved adjuvants, namely, Alum, Immuvac, Montanide ISA720 VG. Enhanced immune response correlated with high IgG antibody titer, Th2 biased response with a high level of rabies virus neutralizing antibodies (RVNAs) and IgG1/IgG2a ratio >1, observed upon alum supplementation of the rabies DNA vaccine. The total IgG antibody titer was 2 IU/ml and total RVNA titer was observed to be 4 IU/ml which is eight times higher than the minimum protective titer recommended by WHO. Furthermore, it conferred 80% protection against challenge with 50 LD₅₀ of the rabies CVS strain, conducted in compliance with the potency test for rabies recommended by the National Institutes of Health (NIH), USA. Previously, we have established pre-clinical safety of this vaccine as per the guidelines of Schedule Y, FDA as well as The European Agency for evaluation of Medicinal Products. The vaccine showed no observable toxicity at the site of injection as well as at systemic level in Wistar rats when administered with 10X recommended dose. Therefore, supplementation of rabies DNA vaccine, pgp.LAMP-1 with alum would lead to development of a non-toxic, efficacious, stable and affordable vaccine that can be used to combat high numbers of fatal rabies infections tormenting developing countries.     

2010年中国狂犬病疫情分析

目的 了解我国狂犬病持续流行相关影响因素,以其对防控工作提供基础数据.方法 采用统计分析和描述流行病学的方法对我国2010年狂犬病的流行情况进行分析.结果 2010年全国23个省817个县（区）报告狂犬病病例为2048例,较2009年下降7.46％.病例以儿童和老人发病率较高,职业以农民（69.14％）为主,男女发病比为2.44：1.全国哨点监测共上报640例个案,致伤动物以犬（87.50％）为主,暴露方式以咬伤为主,病例的暴露后自行处理率、疫苗注射率及被动免疫制剂使用率仍然较差.门诊监测病例暴露后预防处置除个别省外大部分监测点疫苗注射率达98％以上,但Ⅲ度暴露后被动免疫制剂的使用率不高.结论 2010年全国狂犬病疫情有所缓解,门诊病例暴露后预防处置情况良好,但个案病例暴露后预防处置情况依然没有得到改善.     

New approaches to the prevention and eradication of rabies

Despite significant progress in improving the pre- and postexposure prophylaxis of human rabies, the development of better and more cost-effective vaccines and antiviral therapeutics remains a major goal for the treatment of human rabies, the control of animal rabies and particularly for the eradication of rabies virus reservoirs in terrestrial wildlife. In this review, we discuss the structural requirements for an effective rabies vaccine, as well as new strategies currently in use for the development of safer and more potent rabies vaccines for rabies prophylaxis and eradication. Finally, we discuss new immune therapeutics aimed at replacing the conventional administration of antirabies immunoglobulin used in rabies post-exposure prophylaxis in humans.     

Evaluation of tests for rabies antibody and analysis of serum responses after administration of three different types of rabies vaccines.

Humoral antibody response to three types of rabies vaccines were assayed by the neutralization (NT), the mixed hemadsorption (MH), and the indirect immunofluorescence (IF) tests. The NT and MH tests were used to detect antibodies combining with antigens at the surface of virions and infected cells, whereas the indirect IF test measured antibodies mainly to the rabies nucleocapsid antigen. After immunization with a human diploid cell vaccine, antibodies were detected by both the NT and the MH test in the 14th- and 30th-day serum samples from each of eight vaccinated persons. There was a good correlation between titers obtained with the two tests in this group of vaccinees. Antibodies elicited by duck embryo and nervous tissue vaccines occurred less frequently and in lower titers. In these groups of vaccinees, 5 of 14 and 5 of 10, respectively, had antibodies detectable by the NT test in the 14th- and 30th-day sera but were negative by the MH test. It is suggested that this was due to the high levels of immunoglobulin M antibodies, which are known to be elicited by daily injections of vaccine. Since antibodies of the immunoglobulin M class are considered to be less important for protection against rabies, the MH test is recommended for immunity determinations. Compared with the NT test, this test also offers the advantage of being technically more convenient because of its capacity for testing numerous sera in a single run. Antibody titers obtained by the indirect IF test in the human diploid cell vaccine group were relatively low. Titers in the duck embryo and nervous tissue vaccine groups were higher but did not correlate with the results of the NT test.     

24例狂犬病流行病学及临床特点分析

目的 回顾性研究北京地坛医院2008年11月至2010年12月,收治的24例狂犬病例,分析研究狂犬病的流行病学及临床特点,以提高诊治水平,加强狂犬病防治能力.结果 本组病例壮年人群发病多,男性多,农村地区病例多,夏秋季节发病多,潜伏期随暴露部位距头部的距离而延长.36.8％的患者在暴露后处理了伤口,15.8％的患者注射...     

铝佐剂对实验狂犬病疫苗的影响

目的对狂犬病疫苗是否应该含有铝佐剂进行实验研究。方法通过检测疫苗抗体、效力（ＥＤ５０）及建立符合狂犬病现场实际的动物模型比较含铝佐剂和不含铝佐剂疫苗防治狂犬病的效果。结果免疫后第４天和第７天抗体滴度铝佐剂疫苗组明显低于不含铝佐剂疫苗组;在ＮＩＨ效力测定中,铝佐剂疫苗组的ＥＤ５０为９３１３２ｎｇ较疫苗组ＥＤ５０２２１ｎｇ少用抗原量一半左右,但该效力测定法不符合狂犬病现场实际;在符合现场实际的动物模型中,铝佐剂对狂犬病疫苗没有任何促进作用。结论在实验狂犬病中,铝佐剂不促进狂犬病疫苗防治狂犬病,而且有不利影响。