三种ELISA法检测大鼠血清肾综合征出血热病毒特异性抗体的研究

肾综合征出血热(HFRS)是由布尼亚病毒科汉坦病毒属引起的自然疫源性疾病。本病的传染源除黑线姬鼠、褐家鼠、欧洲棕背外,能作为家鼠型或实验动物型HFRS传染源的动物,还有实验大白鼠。近十年来世界各地相继暴发了由实验大鼠引起的HFRS流行,日益引起了人们的高度重视,迫切需要一些快速、简便、敏感、特异的检测手段以用于野生及实验动物的HFRS病毒流行病学监测及检疫,以预防野鼠型、家鼠型及实验室型HFRS的发生。虽然间接免疫荧光试验(IFA)一直是主要可靠的血清学诊断手段,但由于需用昂贵的仪器,并且判定结果有一定的主观性,降低了它的应用价值。我们建立了三种检测实验大鼠体内HFRS病毒抗体的ELISA方法,并对这些方法的敏感性。 (一)取代试验:1.用相应包被液或稀释液代替反应系统中一种或二种成分,结果均为阴性(见表1)。2.以JEV抗原和抗体,HSV抗原和抗体分别取代HFRS病毒抗原和A35McAb、Mark-1 McAb及免抗HFRS病毒免疫血清r-G。以酶标免抗人IgG取代酶标Mark-l,酶标H7 McAb和PAP复合物,羊抗人IgG取代桥抗免抗大鼠血清,结果均为阴性(见表2)。     

SPA—ELISA法检测狂犬病抗体的实验研究

测定狂犬病人或免疫治疗的特异性抗体,是一种评价病人免疫水平的好方法。传统的测定方法是用LD_(50)的实验来完成。但该法较繁琐,需时过长,费用又高,不易被广大患者和实验人员所接受。我们采用了SPA-ELISA法,检测狂犬免疫特异性抗体,实验证明该法简便,重复性好,现报告如下。     

乙型脑炎病毒抗体捕获ELISA检测方法的初步建立

目的 建立检测乙型脑炎病毒抗体的抗体捕获ELISA方法.方法 以乙型脑炎病毒E蛋白抗原表位E39特异的单克隆抗体包被ELISA板,吸附乙型脑炎减毒活疫苗株SA14-14-2病毒粒子,再以吸附的病毒粒子捕获乙型脑炎病毒抗体,建立抗体捕获ELISA方法:同时与以乙型脑炎病毒细胞培养上清包被的间接ELISA方法进行对比,并检测临床血清样品105份.结果 间接ELISA方法背景无法消除,1:10、1:100、1:1000各稀释度的阳性血清与阴性血清吸光度(A)值相近,A阳性血清/阴性血清分别为1.02、0.99、1.13,均<2.1.而抗体捕获ELISA方法1:10、1:100稀释度的A阳性血清/阴性血清分别为3.57、2.94,均>2.1;1:1000稀释度的A阳性血清/阴性血清为1.42,<2.1,表明血清稀释度为1:100时可以显著区别乙型脑炎病毒感染的阳性血清与阴性血清,明显消除间接ELISA方法中背景过高的问题.抗体捕获ELISA方法检测105份临床血清样品,A阳性血清/阴性血清为0.257～0.321(0.262±0.050),均<2.1,为乙型脑炎病毒抗体阴性血清.结论 初步建立了乙型脑炎病毒抗体捕获ELISA方法,该方法特异性较高,对于建立乙型脑炎病毒群的快速鉴别诊断方法具有重要意义.     

Dot—ELISA法和IEST检测旋毛虫抗体的实验研究

旋毛虫病是一种世界性分布的人兽共患病 ,国内屡见报道〔1～ 3〕。确诊主要依据活检 ,因而漏诊、误诊较多。本文采用斑点ＥＬＩＳＡ(Ｄｏｔ -ＥＬＩＳＡ)法和免疫酶染色试验 (ＩＥＳＴ)对感染旋毛虫豚鼠血清进行特异性抗体的检测 ,旨在研究这二种血清学方法的特异性和敏感性 ,以期替代传统的肌肉活检法。1　材料与方法1.1　受试血清　实验感染 7周的旋毛虫病豚鼠血清4 0份 ,每鼠经口感染旋毛虫幼虫 5 0 0条。 4 0只豚鼠实验感染后每周心脏穿刺取血 1次 ,每次 1ｍｌ,连续10周 ,分离血清备用。正常豚鼠血清 4 0份 ,血吸虫病兔血清 2 0份 ,蛔虫…     

乙型脑炎病毒抗体捕获ELISA检测方法的初步建立

Objective To set up an antibody-capture ELISA method to detect the Japanese encephalitis virus(JEV)antibody.Methods ELISA plate was coated with the monoclonal antibody which was specific to the envelope protein epitope E39 of JEV,JEV SA14-14-2 strain as the source of antigen was used to absorb the monoclonal antibody,the absorbed virus used to capture the JEV'S antibody.The antibody that captured ELISA was established.The indirect ELISA method using the virus particles from cell culture was compared with coating ELISA plate,105 clinical serum were checked.Results The background in indirect ELISA assay could not be abscised,positive and negative serum diluted in a ratio of 1:10,1:100,1:1000,the relative value of A posative/A negative were 1.02,0.99,1.13,all<2.1.But the antibody-captured ELISA method when the serum dilution was 1:10,1:100,the A posative/A negative were 3.57,2.94,all>2.1;when the dilution was 1:1000,the A posative/A negative was 1.42,<2.1,it meant the method could distinguish the positive and negative serum efficiently when the dilution Was 1:100,the background problem in indirect ELISA assay could be solved.Antibody-capture method was used to check 105 serum samples,the A posative/A negative over a range of 0.257～0.321(0.262±0.050),all<2.1,no positive sample found.Conclusion The antibody-capture ELISA method has been preliminary set up with a high specificity,capable of quickly identifying JEV from other virus.     

乙型脑炎病毒抗体捕获ELISA检测方法的初步建立

Objective To set up an antibody-capture ELISA method to detect the Japanese encephalitis virus(JEV)antibody.Methods ELISA plate was coated with the monoclonal antibody which was specific to the envelope protein epitope E39 of JEV,JEV SA14-14-2 strain as the source of antigen was used to absorb the monoclonal antibody,the absorbed virus used to capture the JEV'S antibody.The antibody that captured ELISA was established.The indirect ELISA method using the virus particles from cell culture was compared with coating ELISA plate,105 clinical serum were checked.Results The background in indirect ELISA assay could not be abscised,positive and negative serum diluted in a ratio of 1:10,1:100,1:1000,the relative value of A posative/A negative were 1.02,0.99,1.13,all<2.1.But the antibody-captured ELISA method when the serum dilution was 1:10,1:100,the A posative/A negative were 3.57,2.94,all>2.1;when the dilution was 1:1000,the A posative/A negative was 1.42,<2.1,it meant the method could distinguish the positive and negative serum efficiently when the dilution Was 1:100,the background problem in indirect ELISA assay could be solved.Antibody-capture method was used to check 105 serum samples,the A posative/A negative over a range of 0.257～0.321(0.262±0.050),all<2.1,no positive sample found.Conclusion The antibody-capture ELISA method has been preliminary set up with a high specificity,capable of quickly identifying JEV from other virus.     

Sendai Virus and a Unified Model of Mononegavirus RNA Synthesis

Vesicular stomatitis virus (VSV), the founding member of the mononegavirus order (Mononegavirales), was found to be a negative strand RNA virus in the 1960s, and since then the number of such viruses has continually increased with no end in sight. Sendai virus (SeV) was noted soon afterwards due to an outbreak of newborn pneumonitis in Japan whose putative agent was passed in mice, and nowadays this mouse virus is mainly the bane of animal houses and immunologists. However, SeV was important in the study of this class of viruses because, like flu, it grows to high titers in embryonated chicken eggs, facilitating the biochemical characterization of its infection and that of its nucleocapsid, which is very close to that of measles virus (MeV). This review and opinion piece follow SeV as more is known about how various mononegaviruses express their genetic information and carry out their RNA synthesis, and proposes a unified model based on what all MNV have in common.     

流行性出血热患者唾液中特异性IgM抗体检测

本文报道应用IgM—抗体捕捉ELISA法检测流行性出血热患者唾液中特异性IgM抗体的结果。该法特异、敏感。检测出EHF患者发病第一天的唾液IgM抗体,阳性率92.86％(26/28)。其中21例在第15～20病日同时采集血清和唾液标本,检测结果唾液IgM阳性率80.95％(17/21)、将血清唾液IgM抗体均阳性的标本5例经2—ME试验,加热试验证明了其特异性。唾液标本较血清取材更方便、简单、成本低。     

Preparation of Monoclonal Antibodies against the Viral p54 Protein and a Blocking ELISA for Detection of the Antibody against African Swine Fever Virus

African swine fever virus (ASFV) causes a highly contagious viral disease in domestic and wild pigs, leading to serious economic losses. As there are no vaccines or drugs available, early accurate diagnosis and eradiation of infected animals are the most important measures for ASFV prevention and control. Therefore, improvement of available diagnostic assays and development of novel effective techniques are required. This study is devoted to generating a new detection platform of blocking monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) against ASFV p54 protein. Seven monoclonal antibodies against recombinant p54 protein were produced and four epitopes were identified. Three blocking ELISAs were developed with 6A5 and 6F9 mAbs labeled with HRP, respectively, of which the 6A5/6F9-based blocking ELISA displayed the best detection performance, with an AUC of 0.986, sensitivity of 98.36% and specificity of 92.36% in ROC analysis. Moreover, it has an excellent agreement at 96.59% (198/205) when compared to the commercial blocking ELISA (kappa value = 0.920). The method also has high repeatability, with CV <10%, and no cross reaction with the serum antibodies against PRV, PRRSV, CSFV, PCV2 or SVA. This indicates that the 6A5/6F9-based blocking ELISA has high accuracy with good sensitivity and specificity, suitable for viral detection, field surveillance and epidemiological studies.     

ELISA法检测猬迭宫绦虫抗体的研究

目的　探讨研制猬迭宫绦虫特异性诊断方法。　方法　将已克隆的编码猬迭宫绦虫幼虫半胱氨酸酶的基因重组到表达载体内 ,制备高纯度的基因工程抗原 ,以此基因工程抗原制成酶联免疫吸附试剂盒 ,检测 6例裂头蚴病患者血清。　结果与结论　基因工程抗原能与裂头蚴病患者血清发生很强的特异性反应 ,而不能与囊虫病患者血清发生反应。此方法的建立为裂头蚴病的特异性诊断奠定了基础。     

仙台病毒基因结构与功能的研究进展

仙台病毒（SeV）是引起啮齿类动物呼吸道疾病的病原，属于副粘病毒科，单股负链RNA病毒。本文就组成SeV的基因结构和功能做一综述。     

应用双抗体夹心ELISA定量检测鼠疫F1单克隆抗体IgG含量

目的建立一种用于定量检测鼠疫单克隆抗体样品中IgG含量的方法 ,以更好地指导杂交瘤细胞的筛选和抗体的生产、纯化。方法采用羊抗鼠IgG为包被抗体、辣根过氧化物酶标记的羊抗鼠IgG为标记抗体,纯化鼠IgG作参考品,制作标准曲线,测定未知样品中的IgG含量。结果该方法与BCA蛋白浓度测定法测得的结果基本一致,且重复性好,特异性强,灵敏度高。结论双抗体夹心ELISA法可用于测定鼠疫F1单克隆抗体样品中的IgG含量。     

一种间接ELISA检测结果标准化的新方法及其验证

目的建立并验证一种间接酶联免疫吸附法(ELISA)检测结果标准化的方法-改良 ODST法(I-ODST)。方法根据ELISA反应原理推导I-ODST法的公式ODST=αA/1-bA,验证时采用血吸虫抗体检测试剂盒,检测样品中特异性抗体的A值,对A值的倒数与相对浓度C的倒数作线性回归分析,并与常用的A-C、A-logC法进行比较。结果 I-ODST法线性检验回归系数的平方值 (R2)均>0．99,假设检验的P值<0．05,A-C、A-logC法大部分的R2<0．99,且I-ODST法的线性拟合趋势最佳。结论建立了一种比常用数据转换方法线性拟合更好的ELISA检测结果标准化的新方法。     

Development and Validation of an ELISA for the Detection of Bluetongue Virus Serotype 4-Specific Antibodies

In this article, we describe the development and evaluation of a double antigen sandwich enzyme-linked immunosorbent assay (ELISA) able to detect serotype 4-specific antibodies from BTV-4 infected or vaccinated animals using a recombinant BTV-4 VP2 protein. The coding sequence of VP2 was inserted into a pVote plasmid by recombination in the Gateway^® cloning system. Vaccinia virus (VacV) was used as a vector for the expression of the recombinant VP2. After production in BSR cells, recombinant VP2 was purified by immunoprecipitation using a FLAG tag and then used both as the coated ELISA antigen and as the HRP-tagged conjugate. The performance of the ELISA was evaluated with 1186 samples collected from BTV negative, infected or vaccinated animals. The specificity and sensitivity of the BTV-4 ELISA were above the expected standards for the detection of anti-BTV-4 VP2 antibodies in animals reared in Europe or in the Mediterranean basin. Cross-reactions were observed with reference sera for serotypes 10 and 20, and to a lesser extent with serotypes 12, 17 and 24, due to their genetic proximity to serotype 4. Nevertheless, these serotypes have never been detected in Europe and the Mediterranean area. This ELISA, which requires only the production of a recombinant protein, can be used to detect BTV serotype 4-specific antibodies and is therefore an attractive alternative diagnostic method to serum neutralization.     

流行性出血热（EHF）病毒间接ELISA抗原制备的研究

本文报告用硫酸鱼精蛋白结合高浓度盐溶液方法从感染鼠脑组织中提取用于EHF间接ELISA的病毒抗原,对抗原提取条件进行了研究,并成功地将提取的病毒抗原用于检测HFRS病人血清特异性IgG抗体的间接ELISA。