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1.
为了建立蓝舌病(BT)的血清学诊断方法,本研究利用原核表达的蓝舌病病毒(BTV)血清型12型VP7纯化蛋白免疫BALB/c小鼠,制备2株单克隆抗体(MAb),分别命名为BTV-2D10和BTV-4H7。IFA试验表明,2株MAb均能与BTV 24个血清型发生特异性反应,而与茨城病病毒(IBAV)、中山病病毒(CV)、赤羽病病毒(AKAV)、牛病毒性腹泻病毒(BVDV)、牛传染性鼻气管炎病毒(IBRV)、牛轮状病毒(BRV)、牛肠道病毒(BEV)、牛呼肠孤病毒(RV)及口蹄疫病毒(FMDV)无交叉反应,表明2株MAb均为BTV群特异性抗体。采用重组表达的VP7蛋白作为包被抗原建立的竞争ELISA方法证明,BTV-4H7 MAb对不同血清型BTV阳性血清具有良好的阻断效果,而对AKAV、IBAV、BRV和FMDV阳性血清无阻断作用。本研究建立的竞争ELISA方法与IDEXX公司的试剂盒检测包括65份已知背景血清和322份采自广西省的山羊血清样品,检测结果符合率分别达100%和98%。该竞争ELISA方法的建立为BTV抗体的监测提供了安全、快速、准确的技术手段。  相似文献   

2.
为制备抗鸭1型甲肝病毒亚型（DHAV-1a）结构蛋白VP1的单克隆抗体（MAbs）,本研究以pGEX-VP1重组蛋白免疫BALB/c小鼠,同时以纯化浓缩的全病毒作为包被抗原,建立了筛选抗VP1蛋白阳性杂交瘤细胞株的间接ELISA方法,经融合、筛选制备杂交瘤细胞及鉴定MAbs的稳定性、特异性、腹水效价和中和活性等生物学活性,获得了2株持续且稳定分泌抗体的杂交瘤细胞（1A2和5G3）。MAbs腹水ELISA效价分别为1：3.2×104和1：2.0×106。亚类鉴定均为IgG1/κ型。Western blotting结果显示,2株MAbs均能与DHAV-1a VP1蛋白和DHAV-1a全病毒发生特异性反应。特异性试验结果显示2株MAbs能与鸭1型甲肝病毒（DHAV-1）和DHAV-1a发生交叉反应。中和试验结果显示5G3株具有中和活性。结果表明2株MAbs的ELISA效价高、特异性强、稳定性好,均能与DHAV-1和DHAV-1a全病毒发生特异性反应,其中一株具有中和活性。  相似文献   

3.
To prepare the monoclonal antibodies (MAbs) against DHAV-1a, hybridomas were produced by fusing SP2/0 cells with spleen cells from mouse immunized with DHAV-1a pGEX-VP1 recombinant protein. Two hybridomas stablely secreting MAbs against VP1 protein were identified by indirect ELISA detection with DHAV-1a as coating antigen. The ascetic fluids of MAbs were 1:3.2×104 and 1:2.0×106, respectively. The MAbs were IgG1 with κ chain. Western blotting analysis showed that the MAbs could recognize the recombinant VP1 protein and DHAV-1a. The neutralization tests showed that one MAb (5G3) had better neutralization activity. Therefore, the results showed that the ELISA titers and specialities of two MAbs were very good, with excellent stability. In addition, they all had cross interaction with DHAV-1 and DHAV-1a, one of which had good neutralization activity.  相似文献   

4.
为制备蓝舌病病毒(BTV)血清17型VP2蛋白的单克隆抗体(MAb)及鉴定其抗原表位,本研究用原核表达系统部分重叠表达的两段VP2蛋白共同免疫BALB/c小鼠,采用细胞融合技术获得杂交瘤细胞,通过以重组VP2蛋为白包被抗原的间接ELISA筛选获得2株稳定分泌抗BTV17 VP2蛋白的MAbs杂交瘤细胞株,分别命名为3F4和4H10.Ig亚类鉴定2株MAbs均为IgG1/k链.Western blot证明,2株MAbs均能识别重组VP2蛋白.间接免疫荧光试验表明:2株MAbs均与BTV17呈阳性反应,其中MAb 3F4与BTV1、BTV2、BTV3、BTV5、BTV8、BTV11、BTV13、BTV16、BTV23、茨城病病毒(IBAV)、牛轮状病毒(BRV)、牛呼肠孤病毒(RV)均呈阴性反应,但与BTV10和BTV24呈弱阳性反应.利用合成多肽对VP2抗原表位鉴定结果表明,MAb 3F4识别的抗原表位为540DPWNNR545,MAb 4H10识别的抗原表位为540DPWNNRA546.本研究结果为建立BTV17型特异性检测方法及VP2功能研究奠定了基础.  相似文献   

5.
以原核表达纯化的猪(嗜血)支原体MSG1蛋白免疫BALB/c小鼠,运用细胞融合技术筛选分泌针对MSG1蛋白抗体的融合细胞。通过间接ELISA方法筛选获得2株能稳定分泌抗体的融合细胞株,分别命名为1A7和3G6,Western blot结果证明这2株细胞分泌的抗体能够与重组MSG1蛋白发生特异性反应。细胞上清和腹水中的ELISA抗体效价分别为1∶4 096、1∶1 024和1∶1 638 400、1∶51 200,其单抗亚类鉴定均属于IgG1,轻链为κ型。抗原识别位点分析结果表明,2株单抗所识别的抗原位点相同。猪(嗜血)支原体MSG1蛋白特异性单克隆抗体的制备成功,为制备免疫诊断试剂盒和致病机制的研究奠定了基础。  相似文献   

6.
鸡白细胞介素-6原核表达及双抗夹心ELISA方法的建立   总被引：1，自引：0，他引：1  
应用分子生物学技术原核表达ChIL-6，以ChIL-6重组蛋白为免疫原，按免疫程序分别制备兔抗和鼠抗IL-6重组蛋白的多克隆抗体。应用此抗体建立双抗夹心EL-ISA方法后，为了优化此方法本试验对该方法的最佳试验条件、标准曲线、重复性和初步应用进行确定。结果显示，经SDS-PAGE和Western-blot分析，表明ChIL-6在大肠杆菌中正确表达，为了建立检测ChIL6的双抗夹心ELISA方法，本试验应用表达的重组蛋白制备兔抗和鼠抗多克隆抗体。建立的双抗夹心ELISA方法最佳反应条件为包被抗体的质量浓度为50mg／L，4℃过夜；酶标二抗稀释度为1：400，37。C1h；应用该方法检测感染金黄色葡萄球菌后的ChIL-6，其结果与本实验室之前应用人的ELISA试剂盒检测的结果相似。结果表明，本试验建立的检测ChlL-6的双抗夹心ELISA方法可用于临床。  相似文献   

7.
为获得针对猪源EMCV结构蛋白VP2的特异性单克隆抗体,本试验以EMCV结构蛋白VP2为对象,将EMCVBJC3株VP2基因定向插入穿梭质粒中,构建重组腺病毒AdV—VP2,免疫BALB／c小鼠制备单克隆抗体,利用Pepscan技术对单抗识别的抗原位点进行分析。结果显示,间接ELISA及间接免疫荧光法筛选获得了2个阳性细胞克隆株,命名为C11和G8。经检测杂交瘤细胞培养上清抗体效价为1：1600,腹水效价分别为1：2．56×106和1：1.28×106,中和试验鉴定均无中和活性。Western—blot鉴定表明获得的2株单抗均能识别原核表达的重组VP2蛋白。亚类鉴定结果显示2株单抗均为IgG2b亚类,轻链均为κ型。Pepscan分析结果显示,这2株单抗可分别识别EMCVVP2蛋白上的2个B细胞线性表位。本研究获得的蛋白十分接近于其天然构象,很大程度上保证了病毒表面抗原位点的结构和功能。  相似文献   

8.
Colibacillosis accounts for annual multimillion dollar losses in the poultry industry, and control of this disease is hampered by limited understanding of the virulence mechanisms used by avian pathogenic Escherichia coli (APEC). Previous work in our laboratory has found that the presence of the increased serum survival gene (iss) is strongly associated with APEC but not commensal E. coli, making iss and the protein it encodes (Iss) candidate targets of colibacillosis-control procedures. Previously, we produced monoclonal antibodies (MAbs) against Iss to be used as a reagent in studies of APEC virulence and colibacillosis pathogenesis. Unfortunately, the utility of these MAbs was limited because these MAbs exhibited nonspecific binding. It was thought that the lack of specificity might be related to the fact that these MAbs were of the immunoglobulin M (IgM) isotype. In the present study, new MAbs were produced using a different immunization strategy in an effort to generate MAbs of a different isotype. Also, because Iss bears strong similarity to Bor, a lambda-derived protein that occurs commonly among E. coli, MAbs were assessed for their ability to distinguish Iss and Bor. For these studies, the bor gene from an APEC isolate was cloned into an expression vector. The fusion protein expressed from this construct was used to assess the potential of the anti-Iss MAbs produced in the past and present studies to distinguish Bor and Iss. The MAbs produced in this study were of the IgG1 isotype, which appeared to bind more specifically to Iss than previously generated antibodies in certain immunologic procedures. These results suggested that the MAbs generated in this study might prove superior to the previous MAbs as a reagent for study of APEC. However, both MAbs recognized recombinant Iss and Bor, suggesting that any results obtained using anti-Iss MAbs would need to be interpreted with this cross-reactivity in mind.  相似文献   

9.
本研究首先利用同源重组一步克隆法将鸡传染性贫血病病毒(CIAV)的VP3基因克隆到pGEX-6P-1原核表达载体上,经IPTG诱导及SDS-PAGE分析成功获得了重组蛋白rGST-VP3的表达.随即以纯化的重组蛋白rGST-VP3免疫Balb/c小鼠,通过脾细胞与SP2/0细胞融合以及间接免疫荧光(IFA)筛选,获得2株稳定分泌CIAV-VP3抗体的杂交瘤细胞株,分别命名为CIAV-VP3-4D7和CIAV-VP3-4G8;亚型鉴定表明,CIAV-VP3-4D7和CIAV-VP3-4G8均为IgG1;效价测定发现,CIAV-VP3-4D7和CIAV-VP3-4G8的腹水间接免疫荧光效价分别为1:102400与1:12800;Western blot进一步证实,CIAV-VP3-4D7和CIAV-VP3-4G8均能识别重组蛋白rGST-VP3.本研究结果为后期研究VP3蛋白在CIAV致病中作用及其诱导凋亡分子机制奠定了坚实的物质基础.  相似文献   

10.
为制备能够特异性识别牛分枝杆菌（M.bovis）抗酸蛋白酶（MarP）的特异性单克隆抗体（MAb）,本研究利用大肠杆菌表达系统表达了MarP蛋白的胞浆区,并以β-casein为底物、DTT为抑制剂检测MarP的丝氨酸蛋白酶活性。以具有活性的MarP蛋白免疫小鼠,取脾细胞与SP2/0细胞融合,并加入饲养层细胞轻轻摇匀分装于96孔培养板,置于37℃、5% CO2培养箱内培养10 d,通过IFA和间接ELISA筛选阳性细胞株进行亚克隆,制备能分泌针对MarP MAb的杂交瘤细胞株,并通过Western blotting和ELISA检测MAb与重组表达和天然的MarP蛋白的反应性。结果显示,重组表达的MarP具有良好的丝氨酸蛋白酶活性,能够在12 h内将30 μg的β-casein酶解完全,DTT能够抑制MarP的酶活性。具有活性的MarP蛋白免疫小鼠后,获得5株针对MarP的MAb,均能够特异性识别重组和牛分枝杆菌天然表达的MarP蛋白的线性表位,而不与大肠杆菌（E.coli）和耻垢分枝杆菌（M.smegmatis）的蛋白反应。本研究成功制备了MarP蛋白及MAb,为进一步研究MarP的作用底物及其在牛分枝杆菌抗酸胁迫中作用机制提供了必备材料。  相似文献   

11.
非洲猪瘟病毒p62蛋白单克隆抗体的制备及初步应用   总被引：1，自引：1，他引：0  
为制备非洲猪瘟病毒（ASFV）p62蛋白的特异性单克隆抗体,并初步应用于感染组织样品中ASFV抗原的免疫组化（IHC）检测,本研究以杆状病毒表达的非洲猪瘟病毒重组p62蛋白免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞进行融合获得杂交瘤细胞。结果显示：基于纯化的p62蛋白建立的间接ELISA方法对杂交瘤细胞进行筛选和亚克隆,获得了18株可稳定分泌抗非洲猪瘟病毒p62蛋白单克隆抗体的杂交瘤细胞株。经IFA检测,制备的单克隆抗体均与非洲猪瘟病毒反应,且不与猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪圆环病毒2型等猪源常见病毒反应,特异性良好。抗体识别蛋白的鉴定结果显示,3株MAbs识别p35蛋白,15株MAbs识别p15蛋白。14株MAbs重链亚类为IgG1型,4株MAbs重链亚类为IgG2a,轻链均为κ链。利用18株MAbs对ASFV感染猪的肺、扁桃体、淋巴结等组织进行IHC检测,结果显示5株MAbs均能够与感染ASFV的组织发生特异性的免疫反应。本研究获得的非洲猪瘟病毒p62蛋白单克隆抗体可为非洲猪瘟病毒免疫学检测方法的建立及p62蛋白的结构功能等基础研究提供重要的生物材料。  相似文献   

12.
Forty-nine avian chlamydial strains, isolated mainly from various regions in France and from different species of birds, were analyzed and tested with a panel of nine monoclonal antibodies (MAbs) by the indirect microimmunofluorescence test (MIF). The MAbs included five serovar-specific MAbs, three MAbs raised against Chlamydia psittaci and Chlamydia pecorum ovine strains, and one genus-specific MAb. Of the 49 isolates, 41 came from parrots or budgerigars; the rest were from pigeons, a canary, a duck, and a dove. Two additional strains were from unknown hosts. Most of these avian strains were successfully serotyped according to their reactions with five serovar-specific MAbs by the MIF test. The serovars of 44 strains were determined: 39 were of serovar A, 3 of serovar B, and 2 of serovar E. The remaining five isolates were unclassified because they did not react with any of five serovar-specific MAbs but did react with genus MAb or the MAbs produced with ovine strains. The five unclassified isolates (two from budgerigars, two from Gabon gray parrots, and one from a duck) indicate that one or more additional serovars of C. psittaci exist in birds. The heterogeneity within each subgroup was evident because the 49 avian isolates gave 10 subgroups when the results of the five serovar-specific MAbs were combined with results from the three MAbs produced with ovine strains. This heterogeneity of the serovar isolates, as shown by the combination of MAbs, could provide strain markers very useful for epidemiologic studies.  相似文献   

13.
Abstract

Monoclonal antibodies (MAbs) directed against the snakehead rhabdovirus (SHRV) were produced. These MAbs were characterized by immunofluorescence and neutralization tests, and by their ability to immunoprecipitate viral proteins. Of 15 MAbs developed, 9 were isotyped as IgG1 and 6 were IgG2a. Eight of the MAbs recognized the viral glycoprotein in an immuneprecipitation assay. Three of these, designated E1-9A, P10C, and O10F, had neutralizing activity. By immunofluorescence, 12 MAbs showed good binding activity in SHRV-infected epithelioma papulosum cyprini cells. In an indirect fluorescence assay, the MAbs gave varied staining patterns depending upon the viral structural proteins recognized.  相似文献   

14.
Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.  相似文献   

15.
为制备马传染性贫血病毒(EIAV)的单克隆抗体(MAb),本研究以原核表达并纯化的EIAV p26蛋白作为免疫原免疫8周龄的BALB/c小鼠,利用淋巴细胞杂交瘤技术,建立抗EIAV衣壳蛋白p26抗体的杂交瘤细胞.应用重组p26蛋白为抗原建立ELISA筛选方法,获得两株能够稳定分泌特异性MAb的杂交瘤细胞株,分别命名为E11和F8.经试验证明,这些抗p26MAb均能够与感染驴胎皮肤细胞的EIAV和经SDS-PAGE分离的EIAV总蛋白中的p26蛋白结合.这两株p26蛋白MAb的获得将为EIAV的检测及鉴别诊断奠定基础.  相似文献   

16.
The aims of this study were the identification, cloning, and expression of a genetic region encoding an epitope that induces hemagglutination inhibition (HI) antibody against Avibacterium paragallinarum serovar A and an evaluation of the recombinant protein for immunogenicity in chickens. Although two monoclonal antibodies (MAbs) with HI activity, designated S24-951 and S7-1716-5C, were generated in this study, no reactive proteins with both MAbs were identified by Western blot analysis. A gene fragment of 5157 bp, designated hpa5. 1, was cloned from genomic DNA, and a recombinant protein expressed by hpa5.1, designated HPA5.1, reacted with both MAbs on dot-blot analysis. HPA5.1 showed no hemagglutinating activity, but significantly absorbed HI antibodies in the chicken immune serum. Analysis using a series of deletion mutants prepared from hpa5.1 indicated that a 4.8 kbp gene in hpa5.1 is essential for the expression epitope recognized by MAb S24-951. In addition, chickens immunized once with HPA5.1 showed a high protection rate with sufficient HI antibody titers against challenge exposure with a virulent strain of A. paragallinarum serovar A strain 221. These results show that hpa5. I1 is responsible for the expression of an epitope that induces HI antibody, and HPA5.1 might be a candidate for the development of a new vaccine against avian infectious coryza caused by A. paragallinarum serovar A.  相似文献   

17.
A panel of six monoclonal antibodies (MAbs) produced from mice immunized with Pasteurella multocida (M1404) (Heddleston serotype 2) reacted with homologous lipopolysaccharide, as indicated by enzyme immunoassay and immunoblotting. All six MAbs reacted with serotypes 2 and 5 of the 16 Heddleston serotypes. The reactive epitopes were localized on the bacterial cell surface by immunogold labelling. The antibodies could agglutinate P. multocida only if cells were first treated with 1 N HCl. All six MAbs opsonized P. multocida for phagocytosis by mouse macrophages but were not bactericidal in the presence of complement. They afforded only partial protection against infection in mice. The results, together with those of active immunization experiments with LPS, suggest a subordinate role for LPS in protection from experimental infection in mice.  相似文献   

18.
We report on the production and characterisation of monoclonal antibodies (MAbs) against Haemophilus paragallinarum, the causative agent of infectious coryza. A bank of 8 MAbs were produced by traditional techniques - four against the reference strain for Page serovar A (0083) and four against the reference strain for Page serovar C (Modesto). Seven of the eight MAbs were shown to be IgG(1) with one being nontypable. None of the MAbs had HI activity and none gave any detectable reaction when examined by Western blotting. None of the MAbs gave a positive reaction in the indirect ELISA with any of the eight type strains of Pasteurella species or sub-species. None of our 8 MAbs gave serovar specific reactions when used in an indirect ELISA format. There was a trend for the serovar A MAbs to give a higher titre with serovar A isolates/strains and a similar trend for the serovar C MAbs to give higher titres with the serovar C isolates/strains.  相似文献   

19.
Three murine monoclonal antibodies (MAbs) were prepared against an enterotoxin (ET) produced by Bacillus cereus. Although these MAbs were found to react with the ET, their specificities appeared to be different in competitive binding assays. One of the MAbs (D-8), which was highly reactive with the ET, will be useful in developing immunological methods to detect crude ET and to isolate the ET in high yield.  相似文献   

20.
Vaccines for the prevention of fowl pox in chickens and turkeys have been available for more than five decades. However, in recent years outbreaks have occurred in several previously vaccinated chicken flocks. Presumably, fowl poxviruses (FPVs) antigenically different from the attenuated vaccine strains are responsible for such occurrences. In support of this concept, we previously detected minor antigenic changes in field isolates based on comparative immunoblotting with polyclonal anti-FPV serum. Realizing the need for antibodies specific against the dominant antigens of FPV, monoclonal antibodies (MAbs) were produced by immunizing mice with either a field strain of FPV or a pigeon poxvirus, currently used for vaccination. Three hybridoma clones producing MAbs reacting with a specific FPV protein were selected from a total of 83 clones. In immunoblots, two of the MAbs, P1D9 and P2H10, recognized an antigen with an apparent molecular weight varying from 39 to 46 kD, depending on the FPV strain. The third MAb, P2D4, reacted with an approximately 80-kD protein, regardless of which FPV isolate was tested. Immunofluorescent staining with P1D9 and P2D4 revealed that these MAbs react with intracytoplasmic antigens in FPV-infected cells.  相似文献   

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