百合无症病毒单克隆抗体的制备及检测应用

用百合无症病毒(Lilysymptomlessvirus,LSV)免疫的BALBC鼠脾细胞与SP20鼠骨髓瘤细胞融合,经筛选克隆,获得4株能稳定传代并分泌抗LSV单克隆抗体(MAb)的杂交瘤细胞(2A2、5H9、5H2和5E12),并分别制备它们的单抗腹水。4株单克隆抗体腹水间接ELISA效价达10-6,5H9和5E12的抗体类型及亚类均为IgG1,而2A2和5H2均为IgG3,4株单克隆抗体的轻链均为κ链。利用单克隆抗体建立了抗原包被间接ELISA(ACP-ELISA)检测LSV的方法。病叶作1300倍稀释、提纯LSV病毒浓度为18ngmL(每孔的病毒绝对量为1.8ng)时,该方法仍能检测到病毒。利用ACP-ELISA检测了田间样品,发现LSV在百合上发病很普遍。     

芜菁花叶病毒单克隆抗体的制备及检测应用

先以芜菁花叶病毒（TuMV）免疫BAL B/C小鼠,然后取其脾细胞使之与SP2/0鼠骨髓瘤细胞融合,经筛选、克隆,获得4株能稳定传代并分泌抗TuMV单克隆抗体（Mab）的杂交瘤细胞,并以之制备腹水单抗。4株单克隆抗体腹水ELISA效价在10-5～10-6之间,仅对TuMV起特异性反应。Western blot分析表明,4株单抗都能与TuMV 34kD的外壳蛋白亚基起特异反应。利用TuMV的多抗兔血清和单抗腹水建立了三抗体夹心ELISA检测TuMV的方法,检测病叶的灵敏度为1∶5120倍,检测提纯TuMV病毒绝对量为21.9 ng。利用三抗体夹心ELISA测定出7种作物上有TuMV侵染。      

禽网状内皮组织增殖病病毒gp90蛋白单克隆抗体的制备及其识别区分析

为了制备禽网状内皮组织增殖病病毒(REV)gp90蛋白的单克隆抗体,应用His-gp90融合蛋白免疫BALB/c小鼠,取免疫鼠的脾细胞与骨髓瘤细胞(SP2/0)进行融合,经过筛选、3次亚克隆后获得3株稳定分泌抗REV-gp90蛋白的单克隆抗体杂交瘤细胞株,分别命名为3G5-B8、3G5-A10和1G12。经间接ELISA(Enzyme-linked immunosorbent assay)方法检测,单克隆抗体的亲和力解离常数(Kd)分别为6.483×10–10、4.844×10–10和9.330×10–10,3株单抗的亚型分别为Ig G1、Ig G1和Ig G2b。经Western blotting和间接免疫荧光实验检测,3株单抗均能识别REV感染DF-1细胞后产生的gp90蛋白。以Western blotting方法利用单抗检测不同截短的gp90蛋白,初步确定3G5-B8和3G5-A10 2株单抗抗原识别区均位于gp90蛋白第200-245位氨基酸,而1G12株单抗识别区包含第230-235位氨基酸。这些单抗为REV的诊断和致病机理研究奠定了基础。     

中华蜜蜂囊状幼虫病病毒单克隆抗体的制备与鉴定

为了制备中华蜜蜂囊状幼虫病病毒(CSBV)单克隆抗体,本实验利用纯化的中华蜜蜂囊状幼虫病毒(CSBV)免疫Balb/c小鼠,经过3次免疫后,小鼠断尾采血测其血清效价,选择效价高于1∶80 000的小鼠,在细胞融合前3~4d再加强免疫一次。取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,用间接ELISA筛选阳性细胞株并进行3次亚克隆后注射入Balb/c小鼠(Mus musculus)腹腔制备单抗腹水。共获得9株稳定分泌抗CSBV的单克隆抗体,命名为10A1、5B10、5A5、5H2、11D7、9A5、1D3、3C10、10C4,效价都在1∶16 000以上,经间接ELISA实验检测表明,具有较好的特异性。在此基础上,将9株腹水单抗分别与CSBV互作后,接种于2~3日龄中华蜜蜂幼虫,观察幼虫死亡率,研究单克隆抗体中和CSBV病毒能力,结果表明筛选到三株单克隆抗体(10A1、5A5、9A5)对CSBV有中和作用,为CSBV的防治研究奠定了基础。     

牛血清白蛋白单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清白蛋白(BSA)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,培养上清经过双抗体夹心法检测初步筛选分泌鼠IgG的杂交瘤细胞,将此种杂交瘤细胞注射小鼠产生的腹水用间接ELISA法筛选,获得4株能稳定分泌抗BSA单克隆抗体的杂交瘤细胞株,分别命名为2A5、3A3、3G6、4A8。鉴定结果显示,2A5细胞分泌IgG2a/κ,其余3株细胞分泌IgG1/κ;纯化后4株腹水单抗的纯度达90%以上,对BSA的ELISA滴度均可达到1∶100000以上;4株单抗均不与人以及马、猪、羊、兔、豚鼠等血清发生交叉反应;W estern B lotting试验证明4株单抗均识别分子量为68000的BSA;用间接ELISA法测定4株单抗相对亲和力及相对敏感度大小依次为3A3>2A5>3G6>4A8;杂交瘤细胞株连续培养3个月以及冻存半年后复苏,细胞生长良好,杂交瘤细胞分泌的抗体效价稳定。     

蚕豆萎蔫病毒单克隆抗体制备及检测应用

:用蚕豆萎蔫病毒(BBWV)免疫的BALB/C鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得6株能稳定传代并分泌抗BBWV单克隆抗体(Mab)的杂交瘤细胞株,单抗腹水ELISA滴度为1:320000～1:640000,各单抗抗体类型均为IgG1。6株单抗与BBWV不同分离物均有反应,而与其它植物病毒无交叉反应。经Westernblot印迹分析表明,此6株单克隆抗体均是针对BBWV447kD的外壳蛋白大亚基的特异性抗体。这是国内外首次报道获得BBWV单克隆抗体     

巴泰病毒单克隆抗体的制备及鉴定

巴泰病毒(Batai virus,BATV)是一种人兽共患病毒,近年在全球广泛流行。我国也有人和动物感染的相关报道,而目前对BATV的研究仅局限于分子检测和全基因测序,还没有关于BATV单克隆抗体的相关研究,但制备特异性强、活性好的单克隆抗体也是防控BATV的关键。为了制备BATV单克隆抗体,本研究利用纯化的BATV作为抗原免疫雌性BALB/c小鼠,制备鼠源单克隆抗体。通过免疫小鼠后断尾取血,间接ELISA测其血清效价后,将SP2/0细胞与小鼠的脾细胞融合。ELISA方法筛选阳性细胞株,并进行亚克隆后制备单抗腹水,并利用辛酸-硫酸铵沉淀法纯化腹水。结果获得7株单克隆抗体,包括5株IgG型和2株IgM型。其中制备的3E2单克隆抗体效价最高为1∶32 000。经间接ELISA、间接免疫荧光和Western Blot检测表明,制备的3E2单抗具有较好的纯度、活性和特异性,为BATV快速检测方法的建立及致病机制研究奠定了基础。     

抗呼吸道合胞病毒(RSV)单克隆抗体的制备及其对RSV病毒株抗原性的分析

用呼吸道合胞病毒R6(武汉地方株)活毒滴鼻加用戍二醛固定的病毒感染的Hela细胞免疫BALB/C鼠,取脾细胞与小鼠骨髓瘤SP_2/0细胞融合,培育出分泌抗呼吸道合胞病毒单克隆抗体的杂交瘤细胞13株,这些细胞的染色体数为94至104条,其分泌的抗体分别属于鼠IgC_1、IgG_(2a)、IgG_(2b)亚类。腹水荧光抗体滴度为1:10000～1:100000。其中五株单抗有中和病毒作用,尤其是两株中和放价达1:128。应用免疫转印法证实了这些单抗分别能识别RSV6种主要结构蛋白,用7株识别不同病毒结构蛋白的单抗对12株合胞病毒进行抗原性分析,可将这些病毒区分为二个血清型,即A亚型和B亚型。     

口蹄疫病毒单克隆抗体的制备及检测应用

用纯化的口蹄疫病毒(Footandmouthdiseasevirus,FMDV)免疫BALB/C小鼠,将免疫鼠的脾细胞与SP2/0骨髓瘤细胞融合,采用有限稀释法进行克隆,经筛选获得多株能稳定分泌抗FMDV单抗的杂交瘤细胞株。选择其中一株(2G12)用于下列实验,其细胞培养上清液的效价是1:256,腹水效价是1:1280;以自行制备的兔抗FMDV高免血清IgG为捕获抗体包被酶联免疫吸附试验微量反应板,以单抗2G12为第二抗体,建立了快速检测FMDV抗原的双抗体夹心ELISA,该方法能检出90ng病毒,而且只与FMDV发生特异性反应,与猪瘟病毒(HCV)、猪蓝耳病病毒(PRRSV)、伪狂犬病毒(PRV)、猪细小病毒(PPV)和乙脑病毒(JEV)均不发生反应。本研究为检测口蹄疫病毒抗原提供了灵敏和特异的方法。     

单克隆抗体夹心ELISA检测SARS病毒抗原的研究

目的:建立单克隆抗体(McAb)夹心ELISA法,用于检测SARS病毒(SARS-CoV)抗原。方法:用间接夹心ELISA法筛选捕捉和标记用单克隆抗体的组合,采用过碘酸钠法标记辣根过氧化物酶(HRP),优化后用于检测SARS-CoV。结果:从12株抗SARS-CoV鼠单克隆抗体中筛选出2A3/1C5组合用于捕捉SARS-CoV,1A5/1B4组合标记HRP作为指示抗体。优化后2A3/1C5的最适工作浓度为1∶4000,HRP-1A5/1B4的最适工作浓度为1∶2000。本方法检测SARS-CoV的敏感度为105pfu/mL。结论:单克隆抗体夹心ELISA法可特异性检测SARS-CoV抗原。     

猪繁殖与呼吸综合征病毒非结构蛋白NSP2的原核表达及其单克隆抗体的研制

将猪繁殖与呼吸综合征病毒(PRRSV)S1株NSP2蛋白基因进行截短修饰 (tNSP2),克隆于pGEX-6P-1载体,转化大肠杆菌后用IPTG诱导表达。Western-blot结果表明,融合表达的GST-tNSP2蛋白能被PRRSV阳性血清特异性识别,大小约50kD。经GST柱提取纯化蛋白GST- tNSP2,免疫BALB/c小鼠,取脾细胞与SP2/0骨髓瘤细胞进行融合,获得2株能稳定分泌抗NSP2蛋白抗体的杂交瘤细胞株,将其命名为2B5、3H3。亚型鉴定结果均为IgG 1型,其轻链均为Κ链。间接免疫荧光试验证明,2B5和3H3均能与PRRSV S1毒株产生特异性反应,而不能与SY0608毒株反应。从而为PRRSV分离株的鉴定及NSP2功能研究奠定了重要基础。     

新城疫病毒单克隆抗体的特性及应用

建立了8个分泌抗新城疫病毒(NDV)特异性单克隆抗体(McAb)的杂交瘤细胞株,根据它们的免疫生物学特性可以分为三种类型:(1)具有FA和ELISA特性(FN1、FN4、FN29、FN30、FN35、FNl22);(2)具有FA、ELISA和HI特性(FN7);(3)具有ELISA、HI特性和中和能力(FN106),根据FN30和FN106的ELISA试验,可将11个NDV毒株分为二种不同的抗原群,应用FN4-FITC,FN7-FITC和FN29-HRP试剂,对人工感染NDV和野外送检病例检测结果表明,单抗试剂的DFA阳性率(92.3％)高于病毒分离阳性率(87.2％),两种方法的符合率89.7％,这些单抗试剂用于临床诊断敏感性和特异性高,且方法快速、简便。     

抗人α2a型基因工程干扰素单克隆抗体及亲和层析柱的研制

应用鼠－鼠杂交瘤细胞技术建立了三系稳定分泌抗重组人α２ａ型干扰素（ｒＨｕ－ＩＦＮ－α２ａ）单克隆抗体细胞系１Ａ５、１Ｂ５和２７Ａ７。细胞连续传代和在液氮中冻存后复苏,分泌抗体能力不变,并且维持在较高水平,细胞培养上清效价１∶２５６～１∶４０９６,腹水１∶２６×１０５～１∶２７×１０８。Ｉｇ亚类测定,１Ａ５和１Ｂ５ＭｃＡｂ为ＩｇＧ１,２７Ａ７为ＩｇＧ２ａ。三系ＭｃＡｂ均识别ｒＨｕ－ＩＦＮ－α２ａ和－α２ｂ,与ｒＨｕ－ＩＦＮ－α１和正常大肠菌体裂解液无反应。竞争ＥＬＩＳＡ试验,三系ＭｃＡｂ分别针对ｒＨｕ－ＩＦＮ－α２ａ上三个不同表位。同ＭｃＡｂ建立双抗体夹心ＥＬＩＳＡ对ｒＨｕ－ＩＦＮ－α２ａ和－α２ｂ均可检测到１５０ｐｇ／ｍｌ,约１０ＩＵ／ｍｌ的敏感度。用提纯的单抗制备亲和层析柱,单抗偶联率９５％以上。三系单抗亲和层析柱均可将粗制ｒＨｕ－ＩＦＮ－α２ａ提纯到９７％以上纯度。平均回收率为：１Ａ５单抗柱９０２％,１Ｂ５单抗柱９５３％,２７Ａ７单抗柱９４７％。比活性平均值依次为１９４×１０８ＩＵ／ｍｇ,１９７×１０８ＩＵ／ｍｇ和１６４×１０８ＩＵ／ｍｇ,残余鼠ＩｇＧ量均符合规程要求。纯化ｒＨｕ－ＩＦＮ?     

Production of Monoclonal Antibodies to Barley Mild Mosaic Virus and Their Use for Strain Differentiation

A panel of five stable hybridoma cell lines secreting mono- clonal antibodies (mAbs) were produced using a French mechanically transmitted isolate of barley mild mosaic virus (BaMMV-MF) as antigen. All mAbs reacted with BaMMV-MF in two enzyme-linked immunosorbent assay (ELISA) formats: triple antibody sandwich (TAS)-ELISA and antigen-coated plate (ACP)-ELISA. These mAbs recognized epitopes, present on both degraded virions and intact particles. Four mAbs (5C8, 1D5, 1B12, 1A12) belong to the immunoglobulin (Ig)G class and one mAb (3A9) represents an IgM. The five mAbs were compared in TAS- and ACP-ELISA for reactivity with numerous French isolates. These isolates were detected in TAS- and ACP-ELISA with four mAbs (5C8, 1D5, 1B12, 3A9). In both ELISA systems the mAb 1A12 recognized only an epitope specific for BaMMV-MF. All mAbs, except 1A12 recognized also the German (BaMMV-MG), Italian (BaMMV-I) and Japanese (BaMMV-Ka1) isolates in both TAS- and ACP-ELISA. The Japanese isolate (BaMMV-Na1) only reacted with two mAbs (1D5, 5C8) in TAS-ELISA. Only one mAb (3A9) reacted with BaMMV-MF, BaMMV-PF, BaMMV-I,BaMMV-MG and BaMMV-Ka1 in Western blot. These mAbs make it possible to distingish between the three BaMMV serotypes.     

Genome-wide analysis of small RNAs from Odontoglossum ringspot virus and Cymbidium mosaic virus synergistically infecting Phalaenopsis

Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the two most prevalent viruses infecting orchids and causing economic losses worldwide. Mixed infection of CymMV and ORSV could induce intensified symptoms as early at 10 days post-inoculation in inoculated Phalaenopsis amabilis, where CymMV pathogenesis was unilaterally enhanced by ORSV. To reveal the antiviral RNA silencing activity in orchids, we characterized the viral small-interfering RNAs (vsiRNAs) from CymMV and ORSV singly or synergistically infecting P. amabilis. We also temporally classified the inoculated leaf-tip tissues and noninoculated adjacent tissues as late and early stages of infection, respectively. Regardless of early or late stage with single or double infection, CymMV and ORSV vsiRNAs were predominant in 21- and 22-nt sizes, with excess positive polarity and under-represented 5ʹ-guanine. While CymMV vsiRNAs mainly derived from RNA-dependent RNA polymerase-coding regions, ORSV vsiRNAs encompassed the coat protein gene and 3ʹ-untranslated region, with a specific hotspot residing in the 3ʹ-terminal pseudoknot. With double infection, CymMV vsiRNAs increased more than 5-fold in number with increasing virus titres. Most vsiRNA features remained unchanged with double inoculation, but additional ORSV vsiRNA hotspot peaks were prominent. The potential vsiRNA-mediated regulation of the novel targets in double-infected tissues thereby provides a different view of CymMV and ORSV synergism. Hence, temporally profiled vsiRNAs from taxonomically distinct CymMV and ORSV illustrate active antiviral RNA silencing in their natural host, Phalaenopsis, during both early and late stages of infection. Our findings provide insights into offence–defence interactions among CymMV, ORSV and orchids.     

Preparation and characterization of monoclonal antibodies against VP1 protein of foot-and-mouth disease virus O/China99

Monoclonal antibodies (McAbs) 1A9 and 9F12 against Foot-and-mouth disease virus (FMDV) serotype O were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with O/China99. Both McAbs reacted with O/China99 but not with Asia 1, as determined by immunohistochemistry assay. The microneutralization titer of the McAbs 1A9 and 9F12 were 640 and 1 280, respectively. Both McAbs contain kappa light chains, but the McAbs 1A9 and 9F12 were IgG1 and IgM, respectively. In order to define the McAbs binding epitopes, the reactivity of these McAbs against VP1, P20 and P14 were examined using indirect ELISA, the result showed that both McAbs reacted with VP1 and P20. McAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.     

马立克氏病毒单克隆抗体的研究

获得了4株分泌马立克氏病毒(MDV)特异性单克隆抗体(McAb)的杂交瘤细胞:4BS10对MDV所有毒株呈阳性反应;4CN8 对MDV血清1,3型毒株发生反应;2BN90和4CN24只对MDV血清1型毒株有阳性反应。3个McAb属IgG1,1个为IgG2b,均不中和MDV,免疫扩散试验也无沉淀线。对禽白血病毒(ALV)无交叉反应。 以2BN90和辣根过氧化物酶、异硫氰酸荧光素的结合物进行直接酶联免疫吸附试验和直接荧光抗体试验,均获得成功。抗体滴度前者为1/51,200,后者为1/640。对ALV无交叉反应。