犬细小病毒单克隆抗体在犬细小病毒病治疗中的应用

通过选用犬细小病毒单克隆抗体与常规对症治疗两种方法,对206个犬细小病毒病病例,并将其分为两组(每组为103个病例),用两种不同的方法进行对比治疗,其中一组选用了犬细小病毒单克隆抗体对病犬进行治疗,治愈94只,治愈率达91.26%,治疗效果显著高于其它治疗方法。     

犬细小病毒单克隆抗体研究进展

犬细小病毒２型（ＣＰＶ２）是引起犬急性出血性胃肠炎和幼犬急性心肌炎的病原,主要危害幼犬,特别是２～６月龄小犬。该病发病急,病程短,死亡率高,传染性强,危害严重。自Ｅｕｇｓｔｅｒ（１９７７）［１］在美国报道以来,世界各国均有发生和流行［２］。梁士哲等...     

犬及他种动物携带犬细小病毒调查

本文报道采用军事医科院实验中心所生产的犬细小病毒快速诊断试剂检测犬、牛、猪、兔、鸡、鸽、番鸭和樱桃谷鸭８种动物体内携带犬细小病毒，结果发现，除猪以外，其余７种动物均有不同程度的带毒。各种动物的带毒（阳性）比例是：犬为２４／５１、牛为１６／４８、兔为５／５、鸡为５／５、鸽为１０／１０、番鸭为２／１５、樱桃谷鸭为１／１５，在上述带毒动物中，明显出现肠炎症状的只有２只犬，其余均未发现临床症状。     

犬细小病毒单克隆抗体对犬细小病毒病的临床应用研究

采用犬细小病毒单克隆抗体（CPVMcAb）配合静脉注射用犬免疫球蛋白及静脉注射用犬血白蛋白,采用股内侧肌肉注射对172例细小病毒病患犬进行临床治疗试验。共治愈140例,治愈率为81．40％。采取以上治疗方案,结合对症支持治疗,发病1d的患犬治愈率90．70％,发病2d的患犬治愈率为85．29％,发病3d的患犬治愈率为56．25％,发病4d的患犬治愈卒为72．73％,发病5、d的患犬治愈率为77．78％。94．29％的治愈犬（132只）用药后4d内完全康复。采用该治疗方案,进入犬体内的McAb能迅速到达全身各组织,中和淋巴组织及血循环中的病毒,阻止病原复制和扩散;同时,中和反应形成抗原抗体复合物,可促进机体的主动免疫反应,促进患犬逐渐康复。该研究结果对提高犬细小病毒病的临床治疗效果具有积极的应用价值。     

分泌抗犬细小病毒单克隆抗体杂交瘤细胞株的建立

为制备犬细小病毒(CPV)单克隆抗体,用F81细胞繁殖CPV,病毒液经二乙烯亚胺(BEI)灭活、纯化后免疫BALB/c小鼠,将小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,经筛选获得2株分泌抗CPV单克隆抗体的杂交瘤细胞株,命名为F1和B6.F1和B6分泌的单克隆抗体Ig亚类均为IgG1;以F1和B6制备的腹水,经检测,间接ELISA效价均为1×105,免疫荧光效价分别为1:3200和1∶6400;F1和B6培养上清的抗体中和效价分别为1∶1024和1∶2048,血凝抑制效价分别为1∶2048和1∶4096;免疫印迹(Western-blotting)和抗原表位分析结果显示,F1和B6分泌的单克隆抗体可能分别针对CPV的空间表位和线性表位.2株分泌抗CPV单克隆抗体的杂交瘤细胞株的建立可进一步用于开发CPV特异性诊断制剂和治疗制剂.     

犬细小病毒的中西医防治

近些年,养犬的人数不断增加,而犬得病的机率也增加了数倍。犬病中最常见也最需要预防的就是传染病,犬的传染病一般有犬瘟热、犬细小病毒、犬传染性肝炎等,其中发病率最高的是犬细小病毒。一、病原犬细小病毒(CanineParrovirus,CPV)在分类上属于细小病毒科,细小病毒属,CPV可常时     

分泌抗犬细小病毒单克隆抗体杂交瘤细胞株的建立

将用犬细小病毒(CPV)免疫的BALB/C小鼠脾细胞与SP2/0细胞在聚乙二醇作用下融合,用血凝抑制试验(HI)筛选,以有限稀释法克隆3次,得到6株分泌抗CPV单克隆抗体(McAb)的杂交瘤细胞,其中4株分泌IgG_1,2株分泌IgM;6株杂交瘤细胞染色体数目介于96—103之间;在半年传代期间(45代),均能稳定地分泌McAb。将杂交瘤细胞注射BALB/C小鼠诱生腹水,其HI效价介于1:128—1:512之间,置-20℃保存1年,其效价不变。用交又HI法对2株单克隆抗体作特异性分析,该McAb只特异地与CPV发生反应,而不与猫泛白细胞减少症病毒(FPLV)、水貂肠炎病毒(MEV)发生反应。     

Development of monoclonal antibodies against canine glomerular antigens

Monoclonal antibody producing hybridomas were developed by fusing spleen cells from BALB/c mice immunized against canine glomeruli with SP2 myeloma cells. Monoclonal antibody reactivity was tested using an indirect immunofluorescence assay on various normal canine tissues and canine kidney affected with glomerulonephritis. Two of the hybridomas developed (3H2 and 3A5) reacted with glomeruli and not with renal tubules. Antibody produced by hybridoma 3A5 also reacted with smooth muscle of all other tissues tested and 3H2 with lung tissue. Antigens recognized by monoclonal antibodies were studied by assessing their heat stability and susceptibility to proteolysis and neuraminidase digestion. Antigen and antibody molecular weights were determined by using a western blotting technique. Glomerular proteins that reacted with antibody produced by hybridoma 3H2 had molecular weights ranging from approximately 92,500 daltons to 200,000 daltons. Antigens reacting with both monoclonal antibodies were likely protein antigens. It was concluded that monoclonal antibodies would be useful in the study of glomerular antigens in normal dogs and dogs with glomerulonephritis.     

Development and characterization of monoclonal antibodies against canine trypsin

Canine cationic trypsin was purified by salting-out, gel filtration and affinity chromatography. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28 kDa by SDS-PAGE.

Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were purified by salting-out and DEAE ion-change chromatography using ascites fluid of immunized BALB/c mice. The mAb proved to have very high specificity to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays.     

犬细小病毒ELISA抗体检测试剂盒的研制及应用

本研究以纯化的犬细小病毒(CPV)重组VP2蛋白为包被抗原,建立了检测CPV抗体的间接ELISA 方法,其抗原包被浓度为0.158μg/mL;待检血清稀释倍数为1:100,作用时间为60 min;羊抗犬酶标抗体稀释倍数为1:2 000,作用时间为60 min;底物作用时间为10 min;判定标准为S/P≥O.282时为阳性,S/P≤0.226时为阴性.该方法与犬瘟热、犬传染性肝炎、犬副粘病毒病、犬波特氏杆茵痛等4种犬常见传染病阳性血清无交叉反应,具有较好的特异性;批内、批间重复试验变异系数均小于15%,显示该方法具有良好的重复性;与商品化CPV ELISA试剂盒进行比较,符合率为96.5%.本研究为现地免疫犬群抗体监测和进行CPV流行病学调查提供了一种简便的血清学诊断方法.     

Induction and characterization of monoclonal anti-idiotypic antibodies reactive with idiotopes of canine parvovirus neutralizing monoclonal antibodies.

Monoclonal anti-idiotypic (anti-Id) antibodies (Ab2) were generated against idiotypes (Id) of canine parvovirus (CPV) specific monoclonal antibodies (MoAbs). The binding of most of these anti-Id antibodies to their corresponding Id could be inhibited by antigen, thus classifying these anti-Id antibodies as Ab2 gamma or Ab2 beta. By inhibiting experiments it was shown that these anti-Id antibodies did not recognize interspecies cross-reactive idiotopes, but recognized private idiotopes, uniquely associated with the Id of the anti-CPV MoAb used for immunization. This classifies these anti-Id antibodies as non-internal image Ab2 gamma. The potential use of these non-internal image anti-Id antibodies for the induction of antiviral antibodies in the CPV system is discussed.     

犬细小病毒基因疫苗单克隆抗体的制备

为获得可用于犬细小病毒(CPV)感染的治疗和快速诊断的单克隆抗体,从病料中分离了1株犬细小病毒,经PCR扩增VP1全基因并进行序列分析,确定为CPV-2 a型。再将VP1基因克隆到真核表达质粒pc DNA3中,构建了基因疫苗pc DNA3-VP1。用该基因疫苗免疫BALB/c小鼠3次后,取抗体效价高的两只小鼠脾细胞与SP2/0细胞融合,经间接ELISA检测,结果获得了3株杂交瘤细胞株,该3株单抗与犬、猫细小病毒能发生反应,均不与犬瘟热病毒、流感病毒和犬腺病毒发生反应,诱生的腹水可体外中和犬细小病毒,其中两株单抗的中和效价为1∶256,1株单抗腹水的中和效价低于1∶128。结果表明,该CPV VP1基因疫苗可制备具有一定中和效价的CPV单克隆抗体。     

犬瘟热病毒单克隆抗体夹心ELISA检测方法的建立

为建立方便快捷的犬瘟热病毒(CDV)检测方法,本实验应用杂交瘤细胞融合技术,建立了4株分泌抗CDV单克隆抗体(MAb)的杂交瘤细胞株,分别命名为A2、F7、H4和G12.相加ELISA试验显示4株MAb作用于CDV不同的抗原表位.选取相加指数较高的两株MAb G12和A2分别作为捕获抗体和酶标检测抗体,建立检测CDV的夹心ELISA检测方法,并对实验条件进行了优化.结果显示,该方法与犬细小病毒、犬副流感病毒、犬腺病毒1型、犬冠状病毒等犬类病毒无交叉反应,敏感度为5 μg/mL,变异系数小于6％.利用该方法与RT-PCR方法同时检测57份临床样品,两种方法的符合率为100％.本实验建立的MAb夹心ELISA方法具有特异、敏感、方便快捷等优点,适用于大批量临床样品检测.     

Rapid detection of canine parvovirus in feces using monoclonal antibodies and enzyme-linked immunosorbent assay

Monoclonal antibodies were used to develop a double antibody enzyme-linked immunosorbent assay for the detection of canine parvovirus (CPV) antigen in fecal samples. The assay was specific for the hemagglutinating protein of CPV and detected as little as 1.5 ng of virus within a 15-minute incubation period. The use of monoclonal antibodies against 2 epitopes on the CPV antigen permitted the simultaneous addition of test sample and enzyme-conjugated antibody, thus considerably simplifying the manipulations required for the assay. Results were visually determined without special instrumentation. Clinical studies revealed greater than 95% correlation between enzyme-linked immunosorbent assay results and hemagglutination titers.     

Prevalence of antibodies against canine distemper virus and canine parvovirus among foxes and wolves from Spain

Viral diseases can influence the population dynamics of wild carnivores and can have effects on carnivore conservation. Hence, a serologic survey was conducted in an opportunistic sample of 137 foxes (Vulpes vulpes) and 37 wolves (Canis lupus) in Spain for 1997-2007 to detect antibodies against canine distemper virus (CDV) and against canine parvovirus (CPV) by indirect ELISA. Antibodies against CDV were detected in 18.7% of the analyzed animals and antibodies against CPV in 17.2%. There was no difference in antibody prevalence to CDV between both species, even in the same region (P>0.05), but there was a significant difference in antibody prevalence to CPV between foxes (5.1%) and wolves (62.2%) (P<0.05). In fox populations there was a significant difference in antibody prevalence to CDV between geographic areas (Aragón 26.4%, La Mancha 7.8%, P<0.05). In wolf populations there was significantly higher antibody prevalence against CPV (P<0.05) in Castilla y León (100%) than in the Cantabric region (53.3%). There was no significant sex or age-related difference in the antibody prevalence against CDV or CPV in foxes. These results indicate that contact with CDV is widespread among wild canid populations in Spain and that CPV is endemic in the Iberian wolf population. The implications of these results are briefly discussed.     

犬副流感病毒单克隆抗体的研制及其特性鉴定

为研制犬副流感特异性诊断试剂,我们以犬副流感病毒(CPIV)免疫8周龄BALB/c小鼠,采用淋巴细胞杂交瘤技术获得4株稳定分泌针对CPIV的单克隆抗体(MAb)细胞株,分别命名为4F386、584C9、4G7F4和4C9D8.4株MAb腹水针对CPIV的间接ELISA抗体效价达1:10~5～1:10~6,与犬瘟热病毒(CDV)和犬细小病毒(CPV)均不发生交叉反应.MAb 4F386和4C9D8为IgG,5B4C9和4G7F4为IgM.Western blot检测表明,4F386与CPIV的F蛋白发生特异性反应,4G7F4与CPW的HN蛋白发生特异性反应,而584C9和4C9D8不与变性的CPIV蛋白发生反应.4株MAb均具有中和病毒活性,间接免疫荧光检测均呈为阳性.本研究为进一步研制CPIV特异性诊断和治疗制剂创造了条件.     

猪圆环病毒重组Cap蛋白单克隆抗体的制备及初步应用

利用重组杆状病毒表达系统在昆虫细胞中表达PCV2-ORF2基因,并以构建的重组杆状病毒为免疫原,通过杂交瘤技术,研制针对PCV2的单克隆抗体,为建立准确快速的PCV2诊断方法奠定基础。首先将PCV2-ORF2基因克隆到杆状病毒的转移载体pFastBacTM1中,然后将其转化入大肠杆菌感受态细胞DH10Bac中,与DH10Bac中的穿梭载体Bacmid发生转座,通过抗性和蓝白斑筛选,得到重组杆状病毒质粒reBacmid-ORF2,通过脂质体介导reBacmid-ORF2转染Sf9细胞,成功获得了表达PCV2-ORF2基因的P1代重组杆状病毒。将所获得的重组杆状病毒经Vivaflow200浓缩后,免疫6~8周龄BALB/c小鼠,取其脾脏与Sp2/0细胞融合,以IFA进行筛选,经多次亚克隆后得到3株能稳定分泌抗体的杂交瘤细胞,分别命名为5H6、4E2和5F7。通过IFA、IPMA及Western-blot试验对3株单抗特异性进行分析鉴定。结果显示,3株单抗均能与PCV2-Cap蛋白产生特异性的反应,并利用流式细胞术初步建立了对PCV2毒株感染PK15细胞的检测方法,从而为快速检测PCV2病毒奠定了基础。