安徽省两株流行性出血热病毒杂交瘤单克隆抗体性质的比较

两个流行性出血热病毒(EHFV)单克隆抗休(McAb)的性质是不同的,1Hg是血凝抑制抗体,2B_7则不然。前者对14株不同来源EHFV抗原部有免疫荧光反应,后者只对9株起反应,对5株不起反应。这意味着14株EHFV都具有血凝素(HAN)的抗原决定簇,但是1H_8对它呈现不同的活性。     

单克隆抗体对肾综合征出血热病毒50k蛋白的分析

用18株抗肾综合征出血热(Hemorrhagic fever with renal syndrome,以下简称HFRS)病毒McAb,以Western-blot技术分析了纯化的该病毒50k蛋白。结果有7株McAb可与该蛋白反应。这7株McAb的特性(包括感染细胞膜抗原免疫荧光染色模式、中和活性及HI活性等)亦各不相同,提示它们所针对的抗原决定簇的特性也不同。用ELISA阻断试验等证明,上述7株McAb中,有5株所针对的抗原决定簇之间有部分相同或重叠,提示这些具有不同特性的抗原决定簇确实位于同一结构蛋白上。分析结果表明,该50k蛋白的特性及结构均较复杂,尚须进一步研究。     

流行性出血热病毒血凝素抗原位点分析

用八株不同来源的流行性出血热(EHF)病毒的单克隆抗体(McAb),采用血凝抑制试验、反向间接血凝抑制试验、间接酶联免疫及阻断酶联免疫试验等,对两种方法(TE、SA)制备的血凝素抗原进行分析。根据A35、2A6McA5试验的结果,证实血凝素抗原中的核蛋白上存在有非构象依赖性的血凝结合位点;而另外5株McAb在血凝抑制活性方面,虽具有明显的株间交叉,但在反向间接血凝抑制试验、间接酶联免疫试验时则均为阴性,故认为其血凝结合位点位于病毒的膜蛋白,可能与G_2糖蛋白有关,为构象依赖性位点。有关4G6McAb,在阻断酶联免疫试验时,虽与A35、2A6一样具有较高的阻断率,但在反向间接血凝抑制试验、间接酶联免疫试验时明显有别于后二者,对其属性有待进一步确定。     

抗发热伴血小板减少综合征病毒结构蛋白单克隆抗体的制备和功能分析

为制备抗发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)结构蛋白的单克隆抗体,本研究用灭活纯化的SFTSV病毒颗粒免疫BALB/c小鼠,利用杂交瘤技术获得分别分泌抗糖蛋白单抗和核蛋白单抗的杂交瘤细胞株。用免疫荧光法和免疫沉淀方法对制备的单克隆抗体的抗原特异性进行鉴定,并初步进行单抗效价、中和活性及亲和力等功能分析。结果显示,通过细胞融合和克隆化,共筛选出13株稳定分泌抗糖蛋白(Glycoprotein,GP)单抗和7株稳定分泌抗核蛋白(Nucleoprotein,NP)单抗的杂交瘤细胞株。免疫荧光和免疫沉淀鉴定显示获得的单抗有良好的抗原特异性。抗GP单抗中6株针对Gn,7株针对Gc,大部分的间接免疫荧光(Indirect immunofluorescence assay,IFA)滴度在1 280~20 480之间,其中4株抗Gn单抗具有中和活性。获得的7株抗NP单抗均与NP特异性结合,IFA滴度范围在5 120~20 480,均无中和活性。此外,经非竞争ELISA检测的两株抗GP单抗(1C8和1G8)均有较高亲和力。本研究为SFTS诊断方法的发展及SFTSV致病机制研究奠定了基础。     

安徽省流行性出血热病毒杂交瘤单克隆抗体的研制与应用

用流行性出血热病毒(EHFV)李株和陈株血凝素(HAN)分别免疫Balb/C小鼠,取其脾细胞和骨髓瘤细胞SP2/O融合,杂交瘤生长孔率为97.5％和60％,阳性率各为2.5％和22％,克隆化培养阳性率达到100％。通过筛选得2株杂交瘤细胞、一株为2B_7、另一株为1H_8。其腹水单克隆抗体(McAb)滴度均达1∶16×10~4—32×10~4。1H_8的血凝抑制滴度达1∶5120。经染色体核型分析,杂交细胞染色体2B_7为80—97条,1H_8为85—105条,每个细胞均含一条中着丝点标记染色体。McAb的Ig类别和IgG亚类检测结果2B_7为IgM,1H_8为IgG_(2b)。两者对19株不同来源的EHFV具有不同的反应,1H_8McAb与19株都具有不同程度的免疫荧光反应,滴度都较高,且对5株不同来源的EHFV-HAN具有血凝抑制活性,滴度在1∶1280—5120,故1H_8为血凝抑制抗体;2B_7只对12株EHFV起反应,对7株不起反应,且没有血凝抑制活性。这说明1H_8与2B_7具有不同的特性。     

流行性出血热病毒(EHFV)山东地方株特性的研究

本文介绍应用Vero-E_(?)细胞直接从EHF抗原阳性的褐家鼠肺中分离出EHFV-R_(36)株。形态学鉴定符合EHFV,并见到典型的颗粒性EHFV包涵体和大小形态类似的EHFV样颗粒。分离毒株经EHFV单克隆抗体分析,R_(36)株EHFV的抗原谱不同于我国的R_(22)株,它既带有家鼠型R_(22)株相似的抗原决定簇,同时又具有野鼠型抗原决定簇。     

肾综合征出血热病毒糖蛋白的提纯和鉴定

以感染肾综合征出血热病毒(HFRSV)的Vero E6细胞为材料,用免疫亲和层析结合制备聚丙烯酰胺凝胶电泳(PAGE)从感染细胞中提纯了HFRSV两种糖蛋白。先用免疫亲和层析从感染细胞的粗制抗原中获得含有四种蛋白的混合液,用[~3H]-氨基葡萄糖在感染细胞中标记病毒糖蛋白,观察到[~3H]-氨基葡萄糖只结合入78K和57K的病毒蛋白。再用制备SDS-PAGE从HFRSV混合液中提纯78K和57K两种蛋白。实验证明这两种糖蛋白均具中和抗原决定簇,57K的糖蛋白尚具血凝活性,初步鉴定表明这两种糖蛋白相当于文献报道的HFRSV G_1和G_2。     

一株人源抗狂犬病病毒单克隆抗体的表位及中和活性的研究

目的通过ELISA相加试验和狂犬病毒(Rabies virus,RV)逃逸突变试验对一株人源抗狂犬病病毒单克隆抗体(human anti-rabies virus monoclonal antibody,Ab1)进行抗原表位分析。方法将已知表位的对照抗体(CR57和CR4098)和Ab1进行ELISA相加试验;以及对RV逃逸株氨基酸突变位点分析进一步验证Ab1针对的抗原表位,用小鼠中和试验(mouse neutralization test,MNT)分析Ab1的体内抗狂犬病病毒中和活性。结果 Ab1和CR57可同时与RV结合;与CR4098存在竞争,Ab1的逃逸株在氨基酸位点333、336发生突变,MNT检测体内中和活性为550.32 IU/mg,MNT和快速荧光灶抑制试验(rapid fluorescent focus inhibition test,RFFIT)结果基本一致。结论该抗体针对III号抗原表位且具有较高的抗狂犬病病毒中和活性。     

应用单克隆抗体免疫细胞化学法分析尸检组织中肾综合征出血热病毒G2，NP及HA结构蛋白抗原

本文应用15株分别抗肾综合征出血热(HFRS)病毒糖蛋白(Glycoprotein Ⅱ G2),核蛋白(Nuclcocapsid,NP)及血凝素(Hemagglutinin,HA)抗原的单克隆抗体免疫细胞化学方法对19例HFRS尸检病例的16种组织中的病毒抗原进行了定位和分布的研究及抗原分析。结果表明,死于早期HFRS人体组织内病毒抗原量大,分布广泛,主要以可溶性和颗粒性两种形式存在,前者存在于细胞内和细胞外,呈G2,NP及HA抗原阳性,是参与形成免疫复合物的主要抗原形式;后者是以单纯病毒NP或HA抗原阳性的病毒包涵体(IB)形式存在于细胞内,是病毒直接作用所致细胞病变的标志,IB的广泛分布但只有极个别阳性细胞发生坏死,说明该病毒具有泛嗜性感染和弱致细胞病变能力的特性。抗原分析结果显示,组织细胞中病毒抗原的表达及其抗原量受宿主细胞的种属,组织结构特点及病期的影响,也存在个体差异以及因感染病毒株或血清型不同产生差异的可能性。病毒抗原染色形态学证实,NP上具有HA抗原位点,其抗原决定簇有三类,其中的某些抗原决定簇可因病毒宿主动物或细胞的种属不同,表达也不同。HA抗原在人体组织细胞中的高表达和广泛分布也说明HFRSV对人体有很强的侵袭力。     

呼吸道合胞病毒融合抑制活性单克隆抗体的鉴定及应用

用杂交瘤技术制备了抗呼吸道合胞病毒 (RSV Long株 )的单克隆抗体F3细胞株。经IFA及ELISA证明 ,F3株McAb对RSV抗原特异 ,具中和活性及融合抑制活性 ,其腹水中和效价和融合抑制效价分别为 1∶12 8和 1∶6 4。将F3株McAb与商售混合单抗试剂盒进行临床诊断比较 ,二者阳性符合率 96 .9% ,阴性符合率 10 0 %。     

The in vitro and in vivo protective activity of monoclonal antibodies directed against Hantaan virus: potential application for immunotherapy and passive immunization

Hantaan virus (HTNV), a member of the genus Hantavirus, family Bunyaviridae, is an etiologic agent causing a serious human disease, hemorrhagic fever with renal syndrome (HFRS), with a mortality as high as 15% and is also a potential bioterrorism agent. It is urgently needed to develop anti-HTNV-neutralizing monoclonal antibodies (MAbs) for treatment and prevention of HTNV infection. In the present study, 18 murine MAbs directed against HTNV strain Chen were generated and characterized. Among these MAbs, 13 were directed against viral nucleocapsid protein (NP), four recognized the viral envelope glycoprotein G2 and one reacted with both NP and G2. Only those MAbs that recognize the epitopes on G2 were positive in hemagglutination inhibition (HI) test and had in vitro virus-neutralizing activity and in vivo protective activity against HTNV infection of susceptible mice. Since all the mice were protected by administration of the virus-neutralizing MAbs one day before and two days after HTNV challenge, these neutralizing MAbs are potentially useful for pre- and post-exposure prophylaxis and for immunotherapy of HTNV infection. Phase II clinical trials of these neutralizing MAbs for emergent treatment of patients with HTNV infection in early stages of HRFS are carried out in endemic areas in China.     

Topographical analysis of antigenic determinants on envelope glycoprotein V3 (E) of Japanese encephalitis virus, using monoclonal antibodies.

Ten monoclonal antibodies directed against envelope glycoprotein V3 (E) of Japanese encephalitis virus were obtained. They were characterized by hemagglutination inhibition (HI), neutralization, and enzyme-linked immunosorbent assay and divided into four types: flavivirus-cross-reactive HI and non-neutralizing antibody (group 1), subgroup-specific HI and non-neutralizing antibody (group 2), low HI and neutralizing antibody (group 3), and non-HI and neutralizing antibody (groups 4 and 5, respectively). Competitive binding assays were performed to analyze the topography of antigenic determinants by enzyme-linked immunosorbent assay. The results of the competitive binding assay separated non-HI and neutralizing antibody into groups 4 and 5, respectively, and demonstrated the existence of at least five distinct antigenic determinants on V3. The site of group 1 was distinct from any other site. The sites of groups 2 and 3 seemed to be located close together. Our results suggest the following relationship between HI and neutralization: (i) The HI sites are separated from the neutralization sites, and (ii) there are two distinct HI sites, one of which is flavivirus cross-reactive, the other subgroup specific.     

Antigenic and Functional Analyses of Glycoprotein of Rabies Virus Using Monoclonal Antibodies

Thirty-five monoclonal antibodies (MAbs) against glycoprotein (G protein) of the RC-HL strain of the rabies virus have been established. Using these MAbs, two antigenic sites (I and II) were delineated on the G protein of the RC-HL strain in a competitive binding assay. Of these, 34 MAbs recognized the epitopes on site IL Site II was further categorized into 10 subsites according to their patterns in a competitive binding assay. Each site II-specific MAb showed 5 to 23 nonreciprocal competitions. The reactivities of 35 MAbs to rabies and rabies-related viruses in an indirect immunofluorescent antibody test showed that six MAbs in group A binded to rabies and rabies-related viruses and eight MAbs in group E reacted only with rabies viruses, considering that the former represent the genus-specific of Lyssavirus and the latter are rabies virus-specific. From biological assays, 28 of the 35 MAbs showed neutralization activity, 31 showed hemagglutination inhibition (HI) activity, and 18 showed immunolysis (IL) activity. The MAbs recognizing neutralization epitopes fell into at least three groups: those exhibiting both HI and IL activity, those showing only HI activity, and those showing neither HI nor IL activity. All IL epitopes overlap with HA epitopes. Five of the nine MAbs which reacted with the antigen treated by sodium dodecyl sulfate in ELISA were not reduced, or reduced only slightly, in the titer. None of the MAbs reacted with 2-mercaptoethanol-treated antigen. Only one MAb that recognized site I reacted with the denatured G protein in a Western blotting assay, indicating that its epitope is linear. These results suggest that almost all of the epitopes on the G protein of the rabies virus are conformation-dependent and the G protein forms a complicated antigenic structure.     

Antigenic analysis of flaviviruses with monoclonal antibodies against Negishi virus

Twelve monoclonal antibodies against Negishi virus were obtained and characterized by hemagglutination inhibition (HI) and neutralization (NT) tests using five flaviviruses isolated in the pan-Pacific region. The reaction pattern of the antibodies showed that Negishi virus was most closely related to Langat virus, followed by 3-Arch, JE and Apoi viruses in that order. Hemagglutinating (HA) antigen of the virus had distinct HI relating sites which were Negishi virus specific, tick borne encephalitis (TBE) virus complex specific and flavivirus cross-reactive. Monoclonal anti-Negishi antibodies cross-reactive to Japanese encephalitis (JE) virus in the HI test had neutralizing activity to JE virus but no activity to homologous Negishi virus.     

铕螯合剂DTTAEuNa标记流行性出血热单克隆抗体的实验研究

用Ｎ′（对异硫氰基苄基）二乙三胺Ｎ１,Ｎ２,Ｎ３四乙酸铕钠（ＤＴＴＡＥｕＮａ）作为螯合剂,标记不同蛋白浓度的流行性出血热单克隆抗体（ＥＨＦＭｃＡｂ）,经ＳｅｐｈａｋｅｘＧ５０凝胶柱分离标记的单抗分子,通过对层析液的紫外吸收峰处的蛋白光密度,时间分辨荧光强度以及免疫活性测定表明：二批Ｅｕ标记单抗的比活性分别为７８和１４７个Ｅｕ３＋／ＥＨＦＭｃＡｂ,标记回收率分别为４１％和４２％,标记物的免疫活性良好,可用于流行性出血热的抗原及抗体检测     

Properties of monospecific antibodies to the glycoprotein of western equine encephalitis virus

Monospecific (MSp-) antisera against E1 and E2 glycoproteins of western equine encephalitis (WEE) virus were prepared and examined for binding activities to whole virions, hemagglutination-inhibition (HI), neutralization (NT) and protection. Both anti-E1 and anti-E2 MSp-Abs protected mice against WEE virus challenge. A competition experiment with monoclonal antibodies showed that these MSp-antisera appear to lack the antibody population for some epitopes involved in viral neutralization.     

Antibody responses of humans and nonhuman primates to individual antigenic sites of the hemagglutinin-neuraminidase and fusion glycoproteins after primary infection or reinfection with parainfluenza type 3 virus.

An unusual feature of human parainfluenza virus type 3 (PIV3) is ita ability to cause reinfection with high efficiency. The antibody responses of 45 humans and 9 rhesus monkeys to primary infection or subsequent reinfection with PIV3 were examined to identify deficiencies in host immunologic responses that might contribute to the ability of the virus to cause reinfection with high frequency. Antibody responses in serum were tested by using neutralization and hemagglutination inhibition (HI) assays and a monoclonal antibody blocking immunoassay able to detect antibodies to epitopes within six antigenic sites on the PIV3 hemagglutinin-neuraminidase (HN) glycoprotein and eight antigenic sites on the fusion (F) protein. Primary infection of seronegative infants or children with PIV3 stimulated strong and rather uniform HI and neutralizing antibody responses. More than 90% of the individuals developed antibodies to four of the six HN antigenic sites (including three of the four neutralization sites), but the responses to F antigenic sites were of lesser magnitude and varied considerably from person to person. Young infants who possessed maternally derived antibodies in their sera developed lower levels and less frequent HI, neutralizing, and antigenic site-specific responses to the HN and F glycoproteins than did seronegative infants and children. In contrast, children reinfected with PIV3 developed even higher HI and neutralizing antibody responses than those observed during primary infection. Reinfection broadened the HN and F antigenic site-specific responses, but the latter remained relatively restricted. Adults possessed lower levels of HI, neutralizing, and antigenic site-specific antibodies in their sera than did children who had been reinfected, suggesting that these antibodies decay with time. Rhesus monkeys developed more vigorous primary and secondary antibody responses than did humans, but even in these highly responsive animals, response to the F glycoprotein was relatively restricted following primary infection. Bovine PIV3 induced a broader response to human PIV3 in monkeys than was anticipated on the basis of their known relatedness as defined by using monoclonal antibodies to human PIV3. These observations suggest that the restricted antibody responses to multiple antigenic sites on the F glycoprotein in young seronegative infants and children and the decreased responses to both the F and HN glycoproteins in young infants and children with maternally derived antibodies may play a role in the susceptibility of human infants and young children to reinfection with PIV3.     

Characterization of Simian-Human Immunodeficiency Virus Envelope Glycoprotein Epitopes Recognized by Neutralizing Antibodies from Infected Monkeys

We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein epitopes recognized by neutralizing antibodies from monkeys recently infected by molecularly cloned simian-human immunodeficiency virus (SHIV) variants. The early neutralizing antibody response in each infected animal was directed mainly against a single epitope. This primary neutralizing epitope, however, differed among individual monkeys infected by identical viruses. Two such neutralization epitopes were determined by sequences in the V2 and V3 loops of the gp120 envelope glycoprotein, while a third neutralization epitope, apparently discontinuous, was determined by both V2 and V3 sequences. These results indicate that the early neutralizing antibody response in SHIV-infected monkeys is monospecific and directed against epitopes composed of the gp120 V2 and V3 variable loops.     

一株抗H5亚型禽流感病毒血凝素蛋白单克隆抗体的广谱中和活性

以H5N1禽流感病毒株Ck/HK/Yu22/02作为抗原,应用常规杂交瘤技术和血凝抑制实验筛选出抗H5亚型禽流感病毒血凝素蛋白的单抗8H5,单抗8H5经免疫荧光鉴定具有很好的H5特异性.选择33株2002～2006年不同地域,不同宿主中分离的不同遗传变异亚系的H5N1病毒代表株,对单抗8H5分别进行血凝抑制实验及中和试验分析,结果显示单抗8H5对所有H5亚型病毒均有较强反应,而对非H5亚型标准病毒株均不反应,说明8H5是一株广谱性抗H5特异性中和单抗,并提示单抗8H5的HA识别表位可能是一个相当保守的中和表位.并且单抗8H5双抗夹心系统的初步评价显示了其在诊断应用上的前景.