家兔感染EHFV后抗原的分布和抗体应答反应

<正> 近年来,国内外报道了家兔感染流行性出血热病毒(EHFV)后可产生高效价的特异性抗体,朱智勇等首先用家兔作为研究EHFV的易感动物模型,并成功地在动物脏器中找到EHFV抗原。然而,系统地观察动物感染病毒后,抗原在不同脏器中的分布和抗体反应的动态,迄今尚未有详细报道。本文应用EHFV-A9株组织培养抗原,分别在感染前及感染后的第7～20天,用直接和间接免疫荧光技术,检测了兔脾、肝、胰、肺、肾、小肠、大肠等组织切片中的病毒抗原,并在感染后第1～13周内动态观察了血清特异性IgG抗体,现将实验结果报告如     

SARS病毒抗体的检测及其应用

为了解SARS患者血清抗体产生的规律 ,我们用间接免疫荧光法 (IFA)和酶联免疫吸附试验 (ELISA)对广州市第八人民医院的 4 3例住院治疗的SARS患者和 10例正常人的双份血清进行SARS抗体检测 ,同时比较了IFA和ELISA法的特点。10例正常人双份血清的SARS病毒IgM和IgG抗体均阴性。 4 3例SARS患者 ,其中 34例检出IgM占 79.1%。 37例检出IgG占 86 .10 %。SARS病毒IgG抗体的阳性率高于IgM。结果显示 ,发病的同时机体便可检测到SARS抗体IgM最迟离起病时间最长 (33d)的血清也能检测到 ,但也有患者发病第 9、2 3及 30天的第二份…     

两株登革2型病毒E基因重组质粒免疫BALB/c小鼠产生特异性抗体水平的比较

目的 用含登革2型病毒(Dengue type 2 virus,DEN2)B株和NGC株E基因部分序列pcDNA3.1重组质粒初次和加强免疫BALB/c小鼠,观察免疫小鼠体液免疫应答的差异.方法 用两株含DEN2 E基因部分序列(1～476 bp)的pcDNA3.1重组质粒与含有佐剂的重组质粒共同免疫BALB/c小鼠,初次免疫后第14天、28天分别加强免疫1次,共免疫3次.收集初次免疫后第14、28、42、70和98天外周血标本,间接ELISA法测定小鼠血浆特异性IgM/IgG类抗体水平,细胞病变抑制法检测特异性抗体水平.结果 不同DEN2毒株E基因部分序列的pcDNA3.1重组质粒初次和加强免疫BALB/c小鼠诱导特异性IgM、IgG类抗体的产生存在差异,B株重组质粒加强免疫小鼠后特异性抗体效价水平较高并持续较长时间.结论 DEN2两毒株E基因部分序列重组质粒免疫小鼠后诱生的特异性抗体类别、水平存在差异.     

兔抗单克隆个体基因型抗体的制备鉴定

利用具有抑制恶性疟原虫生长能力的94D_1株单克隆抗体(McAb)免疫家兔。得到抗血清后,经Seharose 4 B—正常BALB/c小鼠IgG固相反复吸附3次以上,以除去其抗同种型和同种异型抗体,然后再经Sepharose4 B—9~4D_1株McAb亲和层析柱提纯,得到抗9_4D_1个体基因型抗体。这种抗体经免疫双扩散、IFA封闭试验、SPRIA等方法鉴定,证明具有较高的特异性。在双扩散中,仅与9_4D_1株McAb产生沉淀线;在IFA封闭试验中,能抑制     

Ⅰ型单纯疱疹病毒特异性IgG、IgM及中和抗体的消长动态

本文采用酶联免疫吸附试验(ELISA)间接法、固相抗 IgM ELISA及微量中和试验对 HSV-1感染家兔特异性 IgG、IgM 及中和抗体的消长动态进行观察。结果表明,特异性IgM 在初次感染后第4天可检出,第8～15天达峰值,第66天完全消失,再感染后则检测不出。特异性 IgG 及中和抗体在初次感染后第 8～15天出现,第22天达峰值,检出时间长达 186天以上,再感染后滴度骤升50～100倍。     

肠病毒71型分离株的鉴定及免疫原性研究

目的 在对肠病毒71型(enterovirus 71,EV71)分离株进行鉴定的基础上,对其诱导小鼠产生的免疫应答水平进行研究,拟为进一步的EV71候选疫苗研究奠定基础.方法 超速离心纯化病毒后,采用磷钨酸负染法通过电镜观察病毒形态、大小;采用特异性EV71单抗通过间接免疫荧光法(IFA)检测病毒的特异性;合成EV71特异性引物,通过RT-PCR对病毒进行分子生物学鉴定;病毒的VP1基因序列测定后,与其他EV71序列进行比较,绘制种系发育树,确定病毒基因型;通过腹腔注射途径免疫小鼠,免疫后分离血清通过微量细胞病变抑制法测定血清中和抗体效价,ELISA法检测血清抗体水平和抗体亚型水平.结果 电镜下可观察到典型的圆形病毒颗粒,直径为20～30 nm,呈典型的肠病毒形态;085和087株病毒感染细胞中均能观察到黄绿色荧光,提示该两株病毒可与EV71单抗特异性结合;RT-PCR可以从病毒感染细胞中扩增出226 bp大小的特异性产物带,而采用CA16特异性引物则不能扩增出相应大小的产物带;基于VP1基因序列的种系发育分析显示,087和085株均为C4基因型;085和087株EV71免疫小鼠后可诱导产生能中和包括其本身在内的多个病毒株的中和抗体,针对同一株中和病毒,实验组间差异无统计学意义;针对不同中和病毒株,各实验组中和本株、523-07T株的能力明显高于FY-02T株(P<0.05);ELISA法检测结果显示,接种灭活前后的EV71病毒085和087株后均能诱导小鼠产生抗EV71特异性IgG;IgG亚型为IgG1/IgG2a混合型,灭活病毒免疫组小鼠血清EV71特异性IgG、IgG1、IgG2a水平明显高于活病毒免疫组(P<0.05).两株病毒之间差异无统计学意义.结论 本研究分离到的085和087株病毒均为EV71 C4基因亚型,并具有一定的免疫原性.     

20例SARS患者特异性抗体变化规律

目的：了解严重急性呼吸道综合征(SARS)特异性IgM和IgG抗体的变化规律。方法：采用间接酶联免疫吸附试验(ELISA)检测20例SARS患者系列血清中特异性IgM和IgG抗体，系列血清包括患者发病后1周，2周，3周，4周，8周，12周所采集的样本。结果：20例患者发病后第1周IgM和IgM抗体均为阴性；第2周时16例IgM抗体阳性，17例IgM抗体阳性；第3周后所有患者IgG抗体阳性并持续至第12周，而IgM抗体阳性患者逐渐减少，至第12周时所有患者均为阴性。结论：SARS特异性IgG抗体消失较早，其存在是近期感染的标志；IgG抗体的持续存在可能是获得病后免疫力的标志。     

SARS冠状病毒核壳蛋白单克隆抗体的制备及鉴定

目的制备严重急性呼吸道综合征(SARS)冠状病毒(CoV)核壳(N)蛋白单克隆抗体,为寻求SARS早期诊断的方法及深入研究SARS疾病提供有力的工具。方法以重组SARS—CoVN蛋白免疫小鼠,采用甲基纤维素半固体培养基法制备单克隆抗体。通过间接酶联免疫分析、间接免疫荧光、免疫双向扩散等方法鉴定抗体的特异性、亲和力、类型及亚类、配对效果等。结果共获得15个阳性克隆,其中10个与天然SARS-CoV呈阳性反应。10株单抗的相对亲和常数在108～109mol／L^-1之间;10株中有1株为IgG2b。、1株为IgG3,其余均为IgG1;10株单抗中有8株与12种肺炎相关的病原体无交叉反应,1株与流感病毒A、B,副流感病毒有交叉反应,1株与流感病毒A、B有交叉反应;10株单抗中的5株可形成5种配对,其中两种配对用于检测重组SARS病毒N蛋白,灵敏度可达1μg/L。结论获得了特异性针对SARS冠状病毒N蛋白的单克隆抗体,并初步建立了检测SARS-CoVN蛋白的酶联双抗体夹心法,为SARS的早期诊断、蛋白质组学的研究奠定了基础。     

抗猪流行性腹泻病毒单克隆抗体杂交瘤细胞株的建立及初步应用

用提此的猪流行性腹泻病毒抗原免疫BALB／c小鼠，脾细胞与NS-1骨髓瘤细胞融合，用酶联免疫吸附试验(ELISA)间接法筛选，以有限稀释法克隆化，阳性率选100％，建立了1D8，5D7，4B1，2B3，1A4五株杂交瘸细胞系。用ELISA检测培养上清及诱生的腹水的效价。培养上清效价为1D8 1：1280^x，5D7 1：640^x，4B1 1：320^x 2B8 1：320^x，1A4 1：640^x，腹水效价寿1D8 10^-7，5D7 10^-7、4B1 10^-7，2B8 10^-5，1A4 10^-4。用免疫双扩散试验结果，五株单抗中有一株属IgG类。四株属IgG类。将单抗提此后用ELISA央心间接法及间接免疫荧光试验(IFA)对6神冠状病毒进行了特异性鉴定，除猪流行腹泻病毒外，不与其它冠状病毒发生交叉反应。取效价高而稳定的1D8，5D7，4B1细胞系单克隆抗体做IFA及直接EISA试验初步证明该McAb具有敏感性高，特异性强的优点，可作为猪流行性腹泻病毒检测的一种新试剂。     

检测抗人疱疹病毒6型IgG的间接免疫荧光试验的建立及其应用

目的 :建立检测血清中人疱疹病毒 6型 (HHV 6 )IgG的间接免疫荧光试验 (IFA)。方法 :用HHV 6国内分离株感染人脐带血单个核细胞制备抗原片 ,建立检测HHV 6IgG的IFA方法 ,并用于育龄期妇女血清流行病学调查。结果 :建立的IFA具有特异性。对 116份育龄期妇女血清标本检测表明 ,HHV 6IgG的阳性率为 72 .4 % ,几何平均滴度 (GMT)为 1∶6 1;在孕妇和正常未孕妇女之间 ,以及不同孕期的孕妇之间 ,HHV 6IgG的阳性率和GMT均无差异 (P >0 .0 5 )。结论 :建立了具有特异性的IFA法 ,可用于对育龄妇女HHV 6感染率的流行病学调查     

Reverse ELISA for IgG and IgM antibodies to detect Lassa virus infections in Africa.

BACKGROUND: Anti-Lassa antibodies are detected by indirect immunofluorescence assay (IFA) or by enzyme-immunoassay (ELISA). Both methods have problems to detect low amounts of specific antibodies. OBJECTIVES: We report here highly sensitive and specific reverse ELISAs to detect Lassa virus IgG and IgM antibodies. Due to the reverse techniques, serum samples could be applied at dilutions of 1:10 without increasing non-specific background reactions. STUDY DESIGN: For IgM antibody detection microtiter plates were coated with anti-IgM antibodies and for IgG antibody detection with rheumatoid factor (RF) (Sachers M, Emmerich P, Mohr H, Schmitz H. Simple detection of antibodies to different viruses using rheumatoid factor and enzyme-labelled antigen (ELA). J Virol Methods 1985;10:99-110). In both assays a tissue culture antigen was used in combination with a labeled anti-Lassa monoclonal antibody (Hufert FT, Ludke W, Schmitz H. Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies. Arch Virol 1989;106(3-4):201-12). RESULTS: The reverse ELISA turned out to detect virus-specific IgG and IgM antibody in all 20 samples of West African patients collected 2-8 weeks after onset of Lassa fever. Moreover, both IFA and reverse ELISA found IgG antibodies in 53 out of 643 samples of healthy West Africans (sensitivity of 100%). Six of the 643 samples were positive by reverse IgG ELISA only. Thus, the specificity compared to IIF was 99.0%, but it may be even higher, because compared to IFA the IgG ELISA was clearly more sensitive in detecting low antibody titers. CONCLUSIONS: In Ghana 3% seropositives were found by IFA, but 4% by the reverse ELISA. The reverse ELISAs can be performed with high sensitivity and specificity under field conditions in Africa.     

流行性出血热IgM抗体检测(抗体捕捉ELISA法)

本文报道应用IgM-抗体捕捉酶联免疫吸附法(IgM antibody-capture ELISA,或称反向间接酶联免疫吸附法,Reverse indirect ELISA)检测流行性出血热(Epidemic hemorrhagic fever,EHF)患者血清特异性IgM抗体的结果。该方法能特异、敏感地测出EHF患者发病第2天的血清IgM抗体,阳性率为87.5(7/8),第4病日阳性率达95％(57/60),而病程第7天的38例患者血清全部阳性。15例非EHF发热病人及10例正常人血清无1例阳性,并经2-MB试验与加热试验证明了其特异性。另外,研究表明,对于类风湿因子(RF)干扰所致假阳性反应,本实验所采用的阴性抗原平行对照设计能成功地予以校正而排除。     

Detection of antibody to chicken anaemia agent: a comparison of three serological tests

A comparison was made between sensitivities of the virus neutralisation (VN) test, indirect fluorescent-antibody (IFA) test and enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to chicken anaemia agent (CAA). Sera from chickens inoculated with CAA at 11 weeks of age and from specific pathogen-free (SPF) and field breeder chicken flocks were tested. Seroconversion was detected by the three tests in all the inoculated birds at 2 to 3 weeks post-inoculation (pi). Neutralising antibody to CAA was still detectable in all the inoculated birds 37 weeks after infection, the end of the observation period. One of the seven inoculated birds tested by the ELISA gave positive results for the same period whereas the IFA test detected anti-CAA antibody only for 9 weeks. In field sera, the VN test detected many more positive sera than did the ELISA and IFA test. No antibodies to CAA were detected in sera of SPF chickens by the three tests. The IFA test frequently gave false positive results when VN antibody-negative sera were tested at dilutions of 相似文献   

Immunoperoxidase technique for detection of antibodies to human cytomegalovirus.

The indirect immunoperoxidase antibody technique (IPA) has been applied to determine immunoglobulin (Ig)G to humans cytomegalovirus (CMV) antibodies in 114 blood donor sera, four cases of congenital cytomegalic inclusion disease, and four cases of acquired CMV infection. The results have been compared with those obtained with the CMV complement fixation (CF) test and indirect fluorescent antibody technique (IFA) for broad spectrum CMV antibody (sigmaAb) detection. IgG antibody has been detected by both CF and IPA. In healthy adult people IPA titers are usually higher than CF titers. In addition, IFA sigmaAb titers are generally higher than CF titers. Some sera negative by CF and IPA are positive at low dilutions by IFA sigmaAb antibody determination, due to the detection of small amounts of IgA or noncomplement-fixing IgG. Nonspecific results seem unlikely, since only nuclear inclusion fluorescence was interpreted as specific, as demonstrated by blocking tests. In acute CMV infection, the IFA sigmaAb and IPA IgG titers are essentially the same, except during the first weeks of infection, when IFA titers are higher and IgM is detectable. No cross-reactivity with other herpes group viruses, herpes simplex and varicella-zoster, was observed. Although some problems of nonspecific staining of cytoplasmic inclusions are shared by both methods, the IPA technique seems to possess the same degree of sensitivity and specificity as the IFA technique, but interpretation is easier and various procedural steps can be delayed without the technical problems associated with fluorescence microscopy.     

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。     

Early diagnosis of scrub typhus with a rapid flow assay using recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. We developed a rapid immunochromatographic flow assay (RFA) for the detection of immunoglobulin M (IgM) and IgG antibodies to O. tsutsugamushi. The RFA employs a truncated recombinant 56-kDa protein from the Karp strain as the antigen. The performance of the RFA was evaluated with a panel of 321 sera (serial bleedings of 85 individuals suspected of scrub typhus) which were collected in the Pescadore Islands, Taiwan, from 1976 to 1977. Among these 85 individuals, IgM tests were negative for 7 cases by both RFA and indirect fluorescence assay (IFA) using Karp whole-cell antigen. In 29 cases specific responses were detected by the RFA earlier than by IFA, 44 cases had the same detection time, and 5 cases were detected earlier by IFA than by RFA. For IgG responses, 4 individuals were negative with both methods, 37 cases exhibited earlier detection by RFA than IFA, 42 cases were detected at the same time, and 2 cases were detected earlier by IFA than by RFA. The sensitivities of RFA detection of antibody in sera from confirmed cases were 74 and 86% for IgM and IgG, respectively. When IgM and IgG results were combined, the sensitivity was 89%. A panel of 78 individual sera collected from patients with no evidence of scrub typhus was used to evaluate the specificity of the RFA. The specificities of the RFA were 99% for IgM and 97% for IgG. The sensitivities of IFA were 53 and 73% for IgM and IgG, respectively, and were 78% when the results of IgM and IgG were combined. The RFA test was significantly better than the IFA test for the early detection of antibody to scrub typhus in primary infections, while both tests were equally sensitive with reinfected individuals.     

Serum antibody response to Pseudomonas aeruginosa antigens during corneal infection.

Previous studies in our laboratory have indicated that naturally resistant, inbred DBA/2J mice mount a greater serum antibody response to Pseudomonas aeruginosa 19660 than susceptible C57BL/6J mice. However, the specificity of the antibody produced was not known. The present study examines the specificity and kinetics of the humoral response of these mouse strains to potential virulence factors produced by the organism during both a primary and a secondary corneal infection administered 4 weeks after the primary infection. Serum antibody levels specific for lipopolysaccharide (LPS), exotoxin A, phospholipase C (PLC), alkaline protease, elastase, and flagella were measured by enzyme-linked immunosorbent assay. Little or no antibody to either alkaline protease or elastase was detected during either primary or secondary infection. Immunoglobulin G (IgG) antibodies specific to exotoxin A, PLC, and flagella were detected 2 weeks after primary infection, and a rapid response to these antigens was measured 1 week after secondary infection. During primary infection, detectable LPS-specific antibody was only IgM, while IgG appeared only after secondary infection. The kinetics of the humoral response in susceptible C57BL/6J mice were similar to those in resistant DBA/2J mice, although the magnitude of the response varied according to the antigen tested. These results indicate that LPS, exotoxin A, PLC, and flagella are present or produced in amounts that are immunogenic during corneal infection by P. aeruginosa 19660 in the mouse strains tested.     

Cytomegalovirus-specific antibody responses in renal transplant patients with primary and recurrent CMV infections

Cytomegalovirus (CMV) specific immunoglobulin G (IgG) and immunoglobulin M (IgM) antibody responses were measured before and after renal transplantation in 20 patients with primary CMV infection and in 16 patients with recurrent CMV infection. In primary CMV infection IgG antibody titres to late antigen (IgG-LA) measured by indirect fluorescence (IFA) were approximately seven times higher than those obtained by the complement fixation test (CFT). In contrast, in recurrent CMV infection this difference was found to be about twofold. Virus-specific IgM antibody to late antigen (IgM-LA) was detected in 100 percent of patients with primary CMV infection and in only 50 percent of patients with recurrent CMV infection. The IgM-LA titres were highest in primary CMV infection and reached peak levels at approximately 10 weeks post transplantation, whereas in recurrent CMV infection the IgM-LA titres were lower and reached peak levels at three months post transplantation. Moreover, IgM-LA was found to persist in patients from both groups at nine months post transplantation. IgM antibody to early antigen (IgM-EA) was not detected in any patient in this study. However, significant fourfold titre rises in IgG antibody to EA (IgG-EA) were detected in 100 percent of patients with recurrent CMV infection and in 50 percent of patients with primary CMV infection. These results clearly show the difference in antibody responses to the various antigens of CMV in patients with primary and recurrent CMV infection.     

Enzyme-linked immunosorbent assay for Potomac horse fever disease.

An enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) and IgM in natural and experimental infections of equids with Ehrlichia risticii was developed. Ehrlichial organisms purified from an infected mouse macrophage cell line were used as the antigen. IgM was separated from serum IgG by the expedient of spun-column chromatography, allowing the use of an indirect ELISA for quantitation of both IgG and IgM in the test sera. Among 16 paired sera from horses exhibiting clinical signs of Potomac horse fever, 8 were positive by the indirect fluorescent-antibody test (IFA), 11 were positive by the IgG ELISA, and 8 were positive by the IgM ELISA. All IFA-positive specimens were positive by the IgG ELISA, which appeared to be more sensitive than the IFA. In all cases, the IgG ELISA alone would have sufficed for diagnosis when acute- and convalescent-phase sera were available. When 26 single acute- or convalescent-phase serum samples were tested, the IFA detected 8, the IgG ELISA detected 10, and the IgM ELISA detected 6 positive serum specimens. The kinetics of IgG and IgM responses as determined by ELISA in two experimentally infected ponies which survived infection and challenges revealed that specific IgM was short-lived, falling to undetectable levels by day 60 postinoculation, whereas specific IgG persisted for more than 1 year. IgM and IgG were detected as early as days 1 and 10, respectively, postinoculation. The results suggest that the ELISA is more sensitive than the IFA and that the IgM ELISA may provide a means for early diagnosis of Potomac horse fever at or before the onset of clinical signs.     

不同人群血清SARS冠状病毒抗体检测及其意义

目的　通过对不同人群SARS冠状病毒IgG抗体 (SARSCoVIgG)检测 ,明确该抗体对SARS的诊断意义。方法　采用酶联免疫法 (EIA)、间接荧光法 (IFA)和免疫印迹法 (WB)检测抗 SARSCoVIgG。结果　对 117例临床确诊为SARS患者的 336份系列血清检测表明 ,SARS病人血清抗 SARSCoVIgG最早于发病后第 9天阳转 ,其阳性率随病程延长而上升 ,于发病后 5～ 9、10～ 14、15～ 19、2 0～2 4和 2 5d以上抗 SARSCoVIgG阳性率分别为 12 .5 % (1/8)、73.9% (17/2 3)、91.5 % (43/47)、96 .6 %(5 7/5 9)和 10 0 % (198/198)。应用EIA初筛 12 2 3名非SARS人群 (包括 36 7名在SARS病房工作 1个月以上的医务人员 ,4 3名在生活中与临床确诊的SARS病人有密切接触史者 ,以及 813例未暴露于SARSCoV人群 ) ,其中 2 8名为抗 SARSCoVIgG弱阳性 (A <0 .5 ) ,但用 2种IFA和WB检测均为阴性 ,说明EIA初筛为假阳性。结论　应用EIA检测抗SARSCoVIgG有助于中晚期SARS病人的诊断。对EIA初筛为抗 SARSCoVIgG弱阳性的标本 (A <0 .5 ) ,应用其他方法如IFA和WB检测 ,以排除假阳性。