基于N蛋白单克隆抗体的小反刍兽疫病毒抗体检测胶体金试纸条的研制

本研究旨在建立一种简便、快捷、可直观检测小反刍兽疫病毒(peste des petits ruminants virus,PPRV)抗体的检测方法。将pET-32a-N重组质粒转化至大肠杆菌(Escherichia coli) Rosetta(DE3)感受态细胞中进行诱导表达,以纯化的PPRVN蛋白免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,间接酶联免疫吸附试验(enzyme-linked immunosorbent assays, ELISA)筛选及亚克隆,获得了抗PPRV N蛋白的单克隆抗体。将PPRV N蛋白分别作为金标抗原及检测线(T线)包被抗原、单克隆抗体作为质控线(C线)包被抗体,组装成检测PPRVN蛋白抗体的胶体金免疫层析试纸条。结果显示：成功获得1株能稳定分泌抗N蛋白抗体的杂交瘤细胞株,命名为1F1;间接ELISA检测1F1腹水效价为1:128000;亚类鉴定结果为IgG1,轻链为kappa链。Westernblotting结果显示,1F1能与PPRV N蛋白特异性结合;间接免疫荧光(indirect immunofluorescent ass...     

SARS-CoV单克隆抗体的制备及抗原表位的初步鉴定

参照已发表的SARS冠状病毒BJ01株基因序列 ,利用计算机软件预测并选取该病毒S、M、N三种主要结构蛋白部分抗原性优势区域 ,以编码Gly-Pro-Gly序列相连接合成两段嵌合基因A和B。并分别克隆于pGEX -6p- 1载体上用IPTG进行诱导表达 ,以纯化的嵌合蛋白A和B为抗原 ,分别免疫BALB c小鼠制备单克隆抗体。利用单克隆抗体亚型检测试剂盒和SARS CoV商品化ELISA检测试剂盒对其进行亚型和特异性鉴定。结果表明融合表达两段嵌合基因产物 ,其大小分别为 34kD和35kD ,Westernblot分析证实两种表达产物都能被SARS病人康复期血清所识别。获得了 6株能稳定分泌特异性抗体的阳性细胞克隆株。亚型鉴定结果除D3C5为IgG2a外其他单抗均为IgG1,而且所有单抗的轻链均为κ链。特异性鉴定发现除D3D1外 ,其余的 5株单抗均能与SARS CoV商品化ELISA检测试剂盒发生特异性反应。将D3D1与灭活后经超声波裂解的SARS CoV进行Westernblot分析 ,发现它能特异性识别 180kD的蛋白带。分别融合表达了 6个S蛋白的寡肽 (S1- S6 ) ,并对筛选出的单克隆…     

Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non‐structural Triple Gene Block Protein 1 and Coat Protein

The genes encoding the coat protein (CP) and triple gene block protein 1 (TGBp1) of Potato virus M (PVM) were cloned into expression vector pET‐45b(+) (N‐terminal 6xHis tag) and expressed in E. coli Rosetta gami‐2(DE3). The purified recombinant antigens were used for raising polyclonal antibodies. The antibodies against recombinant CP were successfully used in Western blot analysis, plate‐trapped ELISA and DAS‐ELISA as a coating for PVM detection in infected potato leaf samples. The antibodies against recombinant non‐structural protein detected the TGBp1 only in Western blot analysis. This is the first report of the production of polyclonal antibodies against recombinant coat protein and TGBp1 of PVM and their use for detecting the virus.     

抗猪鼻支原体单克隆抗体的研制及双抗体夹心ELISA检测法的建立

目的 制备多种抗猪鼻支原体的单克隆抗体,建立双抗体夹心ELISA方法用于该病原体的检测。方法用猪鼻支原体CVCC361免疫BALB／c小鼠,采用杂交瘤技术和酶联免疫吸附实验筛选出抗该病原体的单克隆抗体;运用免疫双向扩散试验、Western blotting确定I异G亚类及针对抗原的相对分子质量;筛选出配对抗体,建立双抗体夹心ELISA的检测方法,并评价其灵敏度和特异性。结果共筛选出17株单克隆抗体,抗体亚类分别为IgG1、IgG2a、IgG2b、IgG3,免疫印迹结果表明单抗ZB1、ZB2及ZB16与相对分子质量为35×103的抗原有特异性结合,而ZB3和ZBIO与相对分子质量为70×10^3的抗原有特异性结合。确定了2个配对抗体（ZB1-ZB1-HRP和ZB1-ZB2-HRP）,可检出最小抗原量为30ns／mL,检出猪鼻支原体活菌8．34×10^2CFU／mL,与人呼吸道常见的致病菌及支原体均无非特异性反应。结论筛选的单克隆抗体具有较高的特异性和敏感性,应用双抗体夹心ELISA方法可用于猪鼻支原体的检测。     

抗登革病毒NS1单抗的制备及检测NS1方法的建立

制备抗登革病毒NS1蛋白单克隆抗体,建立检测NS1的ELISA方法。表达1~4型登革病毒NS1蛋白,将1型NS1蛋白纯化后免疫BALB/c小鼠,通过杂交瘤技术制备单克隆抗体。经ELISA、Western blotting、间接免疫荧光筛选和鉴定单克隆抗体,进行纯化和HRP标记。通过鉴定每两株单抗之间是否存在竞争作用,选择非竞争单抗组合并建立NS1捕获法ELISA。结果获得7株高滴度抗NS1单抗,捕获法ELISA可以检出10ng/mL NS1。原核表达登革病毒NS1蛋白制备的单抗可以和天然病毒抗原反应,NS1捕获法ELISA可以用于登革病毒感染检测。     

Characterization and utility of monoclonal antibodies against spike protein of transmissible gastroenteritis virus

Aims: This work aims to characterize the utility of four newly generated monoclonal antibodies (mAbs) against transmissible gastroenteritis virus (TGEV). Methods and Results: Four monoclonal antibodies (mAbs) against the N‐terminal half of spike protein (S1 protein) of TGEV were identified. Affinity constant of these mAbs was analysed. These mAbs were capable of reacting with the TGEV S1 protein analysed by ELISA and Western blot. A competition assay between the different mAbs was performed to determine whether the different antibodies mapped in the same or a different antigenic region of the protein. Investigation on the neutralizing ability of these mAbs indicated that two of these mAbs completely neutralized TGEV at an appropriate concentration. These mAbs were able to detect the TGEV‐infected cells in immunofluorescence assays and Western blot. Moreover, they differentiated TGEV S protein from other control proteins. Conclusions: The generated four mAbs are very specific, and the established immunofluorescence assays, Western blot and discrimination ELISA are useful approaches for detecting of TGEV. Significance and Impact of the Study: It is a novel report regarding the use of the S1 protein of TGEV to generate specific mAbs. Their utility and the established immunoassays contribute to the surveillance of TGE coronavirus.     

Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus

Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D ‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM . About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.     

Effect of Serum Heat-Inactivation and Dilution on Detection of Anti-WNV Antibodies in Mice by West Nile Virus E-protein Microsphere Immunoassay

Immunopathogenesis studies employing West Nile virus (WNV) mice model are important for the development of antivirals and vaccines against WNV. Since antibodies produced in mice early during WNV infection are essential for clearing virus from the periphery, it is important to detect early and persistent anti-WNV antibodies. ELISA and plaque reduction neutralization tests are traditionally used for detection of anti-WNV antibodies and WNV-neutralizing antibodies, respectively. Although these assays are sensitive and specific, they are expensive and time consuming. Microsphere immunoassays (MIA) are sensitive, specific, allow for high throughput, are cost effective, require less time to perform than other methods, and require low serum volumes. Several assay parameters such as serum heat-inactivation (HI) and dilution can alter WNV MIA sensitivity. We examined the effect of these parameters on WNV E-protein MIA (WNV E-MIA) for the enhanced detection of anti-WNV IgM and IgG antibodies. WNV E-MIA was conducted using serial dilutions of HI and non-HI (NHI) serum collected at various time points from mice inoculated with WNV. HI significantly enhanced detection of IgM and IgG antibodies as compared to NHI serum. WNV IgM and IgG antibodies in HI sera were detected earlier at day 3 and IgM antibodies persisted up to day 24 after infection. HI serum at 1∶20 dilution was found to be optimal for detection of both IgM and IgG antibodies as compared to higher-serum dilutions. Further, addition of exogenous complement to the HI serum decreased the WNV E-MIA sensitivity. These results suggest that serum-HI and optimal dilution enhance WNV E-MIA sensitivity by eliminating the complement interference, thereby detecting low-titer anti-WNV antibodies during early and late phases of infection. This improved MIA can also be readily employed for detection of low-titer antibodies for detection of other infectious agents and host proteins.     

利用生物条形码技术对蓝舌病毒VP7蛋白进行微量检测

目的：建立高灵敏检测蓝舌病毒VP7蛋白的生物条形码检测方法。方法：制备VP7蛋白的多抗及特异DNA链标记的金纳米颗粒探针（NP）和VP7蛋白单抗标记的磁性微球探针（MMP）,形成MMP-VP7蛋白-NP三明治复合物后,再利用去杂交将NP探针上标记的DNA链释放出来,通过PCR或芯片检测方法鉴定释放的DNA链,确定VP7蛋白的存在。结果：建立了蓝舌病毒VP7蛋白的生物条形码检测体系,检测灵敏度可达10fg/mL,为常规ELISA检测的106倍。结论：为发展高灵敏度的蓝舌病毒生物条形码检测试剂盒鉴定了基础。     

Preparation and Characterization of a Monoclonal Antibody to Prunus necrotic ringspot virus

A cell line named PVRSV1D11 secreting monoclonal antibody (McAb) against the prokaryotically expressed coat protein (CP) of Prunus necrotic ringspot virus (PNRSV) was developed using hybridoma technology including animal immunization, cell fusion, cell line culture and enzyme‐linked immunosorbent assay (ELISA)‐based for screening. The specificity, titre and detection sensitivity of the McAb were determined by indirect ELISA to establish optimal conditions. The antibody reacted strongly with PNRSV and showed no cross‐reactions with the proteins of Plum pox virus, Prunus dwarf virus, Apple stem pitting virus, Apple stem grooving virus, Apple mosaic virus or Apple chlorotic leafspot virus. The ascites developed with PNRSV1D11 cell line showed high absorbance until it was diluted to over 6.6 × 10⁷ fold. The McAb belonged to IgG_2a isotype and was diluted by 1.28 × 10⁵ folds as an optimal detection concentration. The detection sensitivity of the monoclonal antibody was 11.7 ng/ml protein of PNRSV. The results indicated that the McAb against the CP of PNRSV is suitable for PNRSV detection in the plants and for monitoring the dynamics of the virus by using indirect ELISA.     

Construction and characterization of the chimeric antibody 8C11 to the hepatitis E virus

Homodimers of the truncated hepatitis E virus (HEV) capsid proteins, E2 and p239, were conformed to model the dominant antigenic determinants of HEV. Using E2 as an immunogen, two neutralizing monoclonal antibodies (mAbs), namely 8C11 and 8H3, were produced. We constructed a mouse-human chimeric antibody derived from 8C11 and its expression in Chinese hamster ovary (CHO) cells. cDNAs encoding variable regions of heavy and light chains were isolated from hybridoma cells and inserted into mammalian expression vectors containing cDNA of human gamma-1 and kappa constant regions, respectively. The vectors were then cotransfected into CHO cells, and a stable cell line was established. Results from indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the chimeric antibody was assembled correctly to the native IgG molecule and could be secreted from the cells. Similar to the original mAb, the expressed chimeric antibody displayed HEV antigen-binding activity and an enhancement effect on 8H3 binding to HEV antigen. The chimeric antibody could specifically inhibit the binding of p239 to HepG2 cells and compete with HEV IgG in positive serum by antibody-competitive ELISA. The chimeric antibody is expected to be less immunogenic in human and more suitable for antibody therapy of hepatitis E.     

Detection of white spot syndrome virus (WSSV) of shrimp by means of monoclonal antibodies (MAbs) specific to an envelope protein (28 kDa)

The vp28 gene encoding an envelope protein (28 kDa) of white spot syndrome virus (WSSV) was amplified from WSSV-infected tiger shrimp that originated from Malaysia. Recombinant VP28 protein (r-28) was expressed in Escherichia coli and used as an antigen for preparation of monoclonal antibodies (MAbs). Three murine MAbs (6F6, 6H4 and 9C10) that were screened by r-28 antigen-based enzyme-linked immunosorbent assay (ELISA) were also able to recognize viral VP28 protein as well as r-28 on Western blot. Three non-overlapping epitopes of VP28 protein were determined using the MAbs in competitive ELISA; thus, an antigen-capture ELISA (Ac-ELISA) was developed by virtue of these MAbs. Ac-ELISA can differentiate WSSV-infected shrimp from uninfected shrimp and was further confirmed by a polymerase chain reaction (PCR) and Western blot. Approximately 400 pg of purified WSSV sample and 20 pg of r-28 could be detected by Ac-ELISA, which is comparable in sensitivity to PCR assay but more sensitive than Western blot in the detection of purified virus. Hemolymph and tissue homogenate samples collected from a shrimp farm in Malaysia during December 2000 and July 2001 were also detected by Ac-ELISA and PCR with corroborating results.     

小鼠仙台病毒ELISA抗体检测规范化试剂盒的研制与应用

目的按照实验动物国家标准和农业部对于兽医诊断制品的要求研制小鼠仙台病毒抗体检测试剂盒,并在临床检测中分析其适用性。方法建立仙台病毒的种子批和BHK-21细胞的细胞库;标化仙台病毒生产工艺、抗原蛋白纯化工艺;优化ELISA反应板体系;标化质控血清。使用规范化的ELISA试剂盒对我单位672份送检血清样品进行检测,使用IFA和Western blot方法进行复检。结果病毒的种子批检验表明在-80℃保存半年以上毒力稳定;病毒生产和抗原纯化工艺的标准化提高了抗原生产的稳定性;对照体系的设定降低了环境等变量对于结果判定的影响。在对临床样本的检测过程中发现3种方法的灵敏度ELISA高于Western blot高于IFA。结论规范化的小鼠仙台病毒ELISA抗体检测试剂盒能对小鼠仙台病毒感染状况作出准确判断,具有一定的稳定性和结果可重复性。     

柑橘衰退病毒多克隆和单克隆抗体的制备及检测效果分析

通过改进提纯方法获得了柑橘衰退病毒(Citrustristezavirus,CTV)的提纯液,其产量为1mg/100g植物组织。用CTV免疫大耳白兔,获得多克隆抗体,间接ELISA效价为1∶25600。用CTV免疫小鼠,经细胞融合、ELISA筛选和克隆化培养,获得18株能稳定分泌抗CTV单克隆抗体的杂交瘤单细胞株。对其中4株单克隆腹水抗体进行分析的结果表明,这些抗体的ELISA效价为1∶51200~1∶204800,其中2G和3H的抗体类型及亚类为IgG2a,1E和4H为IgG2b。用所制备抗体对不同来源柑橘样品的CTV检测结果显示,单克隆和多克隆抗体结合使用,采用三抗体夹心ELISA(TAS-ELISA)可以获得理想的检测效果,其特异性强、灵敏度高。同时发现所分析4株单克隆抗体对不同的CTV分离物鉴别能力存在差异,但有关这些CTV分离物的特性及其血清学关系还需进一步研究。     

Detection of three whitefly-transmitted geminiviruses occurring in Europe by tests with heterologous monoclonal antibodies

Selected monoclonal antibodies (MAbs), prepared to particles of African cassava mosaic or Indian cassava mosaic geminiviruses, detected three geminiviruses that occur in Europe: abutilon mosaic virus in Abutilon pictum ‘Thompsonii’, tobacco leaf curl virus in Lonicera japonica var. aureo-reticulata and tomato yellow leaf curl virus in Lycopersicon esculentum. All three viruses were detected in indirect ELISA by MAbs SCR 17 and SCR 20 but they were differentiated by their reactions with SCR 18 and SCR 23. Tobacco leaf curl virus was detected only when reducing agents were included in the leaf extraction medium. Inclusion of sodium sulphite slightly improved detection of tomato yellow leaf curl virus but reducing agents were not needed for detection of abutilon mosaic virus.     

Production and properties of monoclonal antibodies to Solanum nodiflorum mottle sobemovirus

Black raspberry necrosis virus (BRNV) reaches only very low concentrations in herbaceous plants and is difficult to maintain in culture. However, in a mixed culture with an unrelated virus, Solanum nodiflorum mottle (SNMV), in the genus Sobemovirus, the concentration of BRNV particles increases about 1000‐fold. In attempts to produce monoclonal antibodies (MAbs) to BRNV for diagnostic use, purified virus particles from the mixed virus culture were used as immunogen and the resultant antibodies screened against cultures of SNMV alone, BRNV+SNMV and healthy plant extracts. None of the virus‐specific MAbs obtained in this way was specific to BRNV but six were specific to SNMV. Although the original objective was not achieved, the SNMV MAbs were characterised and used to study serological properties of SNMV and other Sobemoviruses. Characterisation of the six SNMV MAbs showed that four were IgG3, one IgG1 and the other IgG2b. SNMV was detected by all six MAbs in ELISA, by five in Western blotting, by three in agarose gel double diffusion tests, but only one was suitable for trapping virus particles in immuno‐electron microscopy (IEM). In Western blotting using virus in sap extracts of Nicotiana clevelandii, each of the five MAbs detected a single major band of Mc. 31 000 in sap containing SNMV, and additional bands of lower mass attributed to degradation of coat protein. In various serological tests, no cross‐reactions were detected between SNMV and seven other viruses from the genus Sobemovirus. However, in IEM but not in Western blotting, significant cross‐reactions were observed between SNMV and Velvet tobacco mottle virus, another species from the genus Sobemovirus. The significance of these different findings is discussed.     

蝶蛹金小蜂卵黄蛋白单克隆抗体的制备及其应用方法的建立

为深入研究寄生蜂卵黄发生及其内分泌调控,特采用杂交瘤细胞技术,制备4株能稳定分泌抗蝶蛹金小蜂Pteromalus puparum卵黄蛋白（vitellin, Vt）的单克隆抗体（mAb）,即PpVt mAb1,PpVt mAb2,PpVt mAb3和PpVt mAb4。这4株单克隆抗体的重链和轻链的亚类均分别为IgG1和κ类型,不仅特异性识别Vt,而且识别雌蜂血淋巴中卵黄原蛋白（vitellogenin,Vg）,但与雄蜂体液无反应。通过比较4种不同的ELISA方法,确定了微量检测蝶蛹金小蜂体内Vg/Vt的最适ELISA法,即双夹心ELISA法。该方法可用于单头雌蜂体内Vg/Vt的检测,其检测灵敏度为20 ng/mL。用Western 免疫印迹的方法证实了该蜂Vg的合成始于刚羽化的成虫,并在羽化后12～36 h内含量达到高峰。     

抗胆汁螺杆菌单克隆抗体的制备

目的制备抗胆汁螺杆菌单克隆抗体（McAbs）。方法用胆汁螺杆菌B2m株皮下免疫BALB/c小鼠,采用杂交瘤技术进行融合。以酶联免疫吸附实验（ELISA）筛选抗胆汁螺杆菌单克隆杂交瘤细胞株并初步鉴定其特异性;免疫印迹试验测定单抗所结合的抗原表位;免疫双向扩散试验确定IgG亚类;腹腔接种法、辛酸-硫酸铵盐析法大量制备、纯化单克隆抗体。结果经过ELISA筛选获得11个阳性杂交瘤细胞株,其效价最高达1：4×10^5以上,并与实验动物常见的15种病原菌呈阴性反应;IgG亚类为IgG2a和IgG2b;免疫印迹试验显示,6株（A-F）与胆汁螺杆菌大约相对分子质量（172、0、21、30、52、66）×10^3的抗原特异结合,5株（G-K）皆与胆汁螺杆菌、幽门螺杆菌等三种螺杆菌大约相对分子质量（52、82）×10^3的抗原呈阳性反应,表明A-F株针对的是胆汁螺杆菌特异性抗原,G-K株可能具有属特异性。结论筛选的单克隆抗体具有较高的特异性和敏感性,所结合的抗原为胆汁螺杆菌或螺杆菌的免疫优势抗原,为进一步的种、属生物学特性研究、菌株分型及血清学检测方法建立等奠定了基础。