Characterization of stage-specific and cross-reactive antigens from Eimeria acervulina by chicken monoclonal antibodies

The characterization of five chicken monoclonal antibodies (mAbs) that were developed against apical complex antigens of Eimeria acervulina sporozoites is realized and the mAbs reactivity to merozoites belonging to this species is tested. Using immuno-fluorescence assay (IFA), one mAb (HE-4) that recognized apical antigens common to sporozoites of E. acervulina and E. brunetti bound antigens localized on the apical tip of merozoites from all stages of development examined. The mAb 8E-1, reactive with antigens found on the apical tip of all chicken Eimeria sporozoites, also showed binding to antigens common to merozoites from all generations. Another mAb, 8C-3, which identified an antigen shared by sporozoites apical tip and sporocysts wall of E. acervulina reacted very weak and inconstantly with the merozoites from all generations whereas the mAbs 5D-11 and 8D-2 that recognized antigens shared by the sporozoites of E. acervulina and E. maxima (mAb 5D-11) and E. acervulina and E. brunetti (mAb 8D-2) did not react with the merozoites from any generation. Collectively, these results showed that the invasive stages of chicken Eimeria share cross reactive apical complex antigens which are inter-species and inter-generation-specific that might be components of a potential recombinant vaccine.     

两株抗猪伪狂犬病毒的单克隆抗体制备与鉴定

采用猪伪狂犬病毒(PRV)ZJ01株纯化病毒免疫BALB/c小鼠,利用融合细胞技术和间接ELISA抗体筛选技术,制备并获得2株能稳定分泌抗PRV单克隆抗体的杂交瘤细胞株2B3和5C10,其中,2B3单抗为Ig G2a亚类,5C10为Ig G1亚类,轻链均为κ链。间接免疫荧光检测结果表明,2株单克隆抗体均能与PRV发生特异性反应。Western-blot结果表明,2B3单抗针对PRV g C蛋白,5C10单抗针对PRV g E蛋白。本研究为建立快速检测伪狂犬病毒感染的免疫学方法奠定了基础。     

猪伪狂犬病病毒变异毒株gE和gB蛋白单克隆抗体的制备与鉴定

为建立猪伪狂犬病病毒(PRV)疫苗免疫抗体与病毒感染抗体鉴别诊断方法,分别以PRV变异毒株的gE或gB重组蛋白免疫BALB/c小鼠,将免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,经免疫过氧化物酶单层细胞试验(IPMA)和酶联免疫吸附试验(ELISA)筛选所需单克隆抗体,获得6株抗PRV gE蛋白的单抗(3E6、7E1、7C5、10C3、15C1、14C1)和6株抗PRV gB蛋白的单抗(1C1、5D2、2F11、5G8、10C7、8H5)。IPMA检测结果显示,gE蛋白单抗能识别PRV变异毒株,但不识别疫苗株;而gB蛋白单抗可同时识别PRV变异毒株和疫苗株。gE单抗10C3和gB单抗10C7的ELISA效价和IPMA效价均分别达1∶5×10~(5.0)和1∶8×10~(3.0)以上。所筛选出的单抗均不与猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪流行性腹泻病毒(PEDV)、流行性乙型脑炎病毒(JEV)等其他常见病毒发生交叉反应。gE蛋白和gB蛋白的表达及单抗的制备为PRV感染抗体与疫苗免疫抗体鉴别诊断(DIVA)试纸的研发奠定基础。     

猪伪狂犬病毒gE蛋白单克隆抗体制备与鉴定

为获得猪伪狂犬病毒(PRV)gE蛋白单克隆抗体,选择原核表达的重组gE蛋白免疫6周龄BALB/c雌性小鼠,将其脾细胞与SP2/0进行融合,经间接ELISA筛选阳性杂交瘤细胞,结果获得了2株能稳定分泌抗PRV gE蛋白的杂交瘤细胞,命名为E3B8和E5C11。间接ELISA检测2株杂交瘤细胞的培养上清液抗体效价为1∶6.4×10~3,腹水的抗体效价分别达到1∶3.28×10~6和1∶6.55×10~6。2株杂交瘤细胞的染色体数分别为105和108。E3B8亚类鉴定重链为IgG1,轻链为κ链;E5C11亚类鉴定重链为IgG2b,轻链为κ链。Western blot检测显示2株单克隆抗体腹水均能与PRV重组gE蛋白发生特异性反应,间接免疫荧光试验(IFA)检测显示2株单克隆抗体均能与PRV分离毒株感染的BHK-21细胞发生特异性反应,交叉反应性检测显示2株单克隆抗体与常见病毒不发生交叉反应。表明制备的2株gE蛋白单克隆抗体效价高、特异性强,为gE蛋白结构与功能分析以及PRV免疫诊断试剂盒的开发奠定了基础。     

分泌抗猪伪狂犬病病毒(北京株)单克隆抗体杂交瘤细胞株的建立

用纯化的猪伪狂犬病病毒免疫Ｂａｌｂ／ｃ小鼠,取脾细胞与ＳＰ２／０骨髓瘤细胞融合 ,经间接ＥＬＩＳＡ筛选,３次有限稀释法克隆,获得了２株能稳定分泌抗伪狂犬病病毒单克隆抗体杂交瘤细胞株１Ｈ７和３Ｂ５,经鉴定两株单抗均为ＩｇＧ１亚类、Ｋａｐｐａ型轻链,杂交瘤细胞的平均染色体数为９７,细胞培养液上清及腹水效价分别为１：１０２４、１：１０２４和１：１０＾８、１：１０＾７;１Ｈ７、３Ｂ５单克隆抗体不与猪繁殖－呼吸综合征病毒、猪温病毒、猪细小病毒发生交叉反应,显示良好的特异性,为进一步应用奠定了基础。     

Analysis of swinepox virus antigens using monoclonal antibodies.

Seventeen monoclonal antibodies (MAbs) against swinepox virus (SPV) were produced and characterized. These MAbs were classified into eight groups (A through H) on the basis of the molecular weight of the polypeptides which they recognized and the staining patterns of antigens in SPV-infected cells by the indirect immunofluorescent (IF) technique. The MAbs belonging to groups A, B, C and G recognized late antigens in cytoplasmic inclusion bodies with molecular weights of 97 kD, 65 kD, 48 kD and 15 kD, respectively. The MAbs belonging to groups D and H respectively recognized 35 kD and 12 kD late antigens, which first appeared in cytoplasmic inclusion bodies and spread to the cytoplasms and surface membranes of the infected cells. The MAb of group F recognized an 18 kD late antigen with granular distribution in the cytoplasm. The MAbs of group E recognized a 32 kD early antigen. Although all the MAbs belonging to the six groups (A, D through H) were specific for SPV, some of those belonging to groups B and C showed cross-reactivity with members of the other genera of poxviridae. An MAb in group B, SP14, cross-reacted with orf and rabbit fibroma viruses. Two MAbs in group C, SP24 and SP32, cross-reacted with vaccinia, cowpox, ectromelia, and rabbit fibroma viruses. These findings indicate that at least two SPV antigens contain cross-reactive epitopes with different genera of poxviridae.     

Identification of virus-specific antigens in cultured cells infected with Marek's disease virus serotype 2 and CVI-988 strain

For the identification of serotype-specific antigens of Marek's disease virus (MDV) serotype 1 (MDV1) or serotype 2 (MDV2), a total of 24 hybridoma clones, secreting monoclonal antibodies (MAbs) against CVI-988 (MDV1) or HPRS-24 (MDV2) strain, were established and characterized by immunofluorescence assay, virus neutralization and immunoprecipitation analysis. Based upon the molecular weights (mol. wt.) of the immunoprecipitated polypeptides, the MAbs were subdivided into 7 groups. Among them, two groups of MAbs reacted with antigens that have not been reported, were identified. MAbs belonging to the first group reacted with CVI-988- and MDV2-specific antigens with mol. wt. ranging from 29 K to 34 K (29/34 K). This antigen was not found in cells infected with Md/5 and JM strains of MDV1, and the results of kinetic analysis of antigen expression showed this antigen appeared to be related to late membrane antigens. MAbs belonging to the second group immunoprecipitated MDV2-specific antigens with mol. wt. of 37 K, 33 K and 31 K from HPRS-24-infected cells or with those of 37 K, 34 K and 31 K from SB-1(MDV2)-infected cells, and these antigens appeared to be related to early antigens. MAbs belonging to the other 5 groups included those which recognized similar antigens reported previously or the antigens characterized insufficiently in this study.     

Persistence of inclusions and antigens of chicken anemia virus in Marek's disease lymphoma

The pathogenesis of the co-infection of CAV to MDV is complicated. In order to investigate the impact of CAV on the transformation phase of MD, MDV and, subsequently, CAV, were inoculated at 1day and 4weeks of age, respectively. Chickens were divided into six groups; vvMDV, vvMDV-CAV, vMDV, vMDV-CAV, CAV and a control group. The CAV inclusions and antigens were continuously detected in MD lymphomas in the vMDV-CAV and vvMDV-CAV groups in large bizarre-shape (presumably CD4(+) T cells) and small MD lymphoid cells (presumably CD8(+) T cells). The MD lymphomas were composed primarily of CD4(+) T cells, but CD8(+) T cells were infiltrated singly or in clusters. CAV enhanced the MDV-induced brain lesions in the vMDV-CAV group. The lymphoproliferative lesion (LP) in the vvMDV-CAV and vMDV-CAV groups was non-significantly higher than those in vvMDV and vMDV groups, respectively. CAV significantly increased the LP lesion in sciatic nerves. In conclusion, MD lymphomas enabled CAV replication and dissemination. The depletion of CTLs by CAV did not significantly affect progression of MD lymphoma, although they are essential for possible transition of lymphomatous to inflammatory lesion.     

Protection of mice and swine from pseudorabies virus-induced mortality by administration of pseudorabies virus-specific mouse monoclonal antibodies

Hybridomas were selected for secretion of monoclonal antibodies directed against pseudorabies virus (PRV) glycoproteins. Each monoclonal antibody was capable of neutralizing PRV in vitro in the presence of complement. This panel of antibodies was used in passive immunization studies to protect mice and swine from PRV-induced mortality. The most protective antibody in mice was 3A4, specific for PRV glycoprotein gp50, which afforded as high as 100% protection. Although antibody 3A4 was partially protective in swine, antibody 3D11, which is specific for PRV glycoprotein glll, afforded greater protection--83% protection when ascitic fluid was used and 100% protection when immunoglobulin concentrated from cell cultures was used at a dose of 150 mg/pig. These studies demonstrated that monoclonal antibodies may be useful for short-term prophylaxis against PRV-induced disease and that antibody directed against either PRV glycoprotein glll or gp50 is sufficient to protect animals from PRV-induced mortality.     

Borna disease virus-specific antigens: two different proteins identified by monoclonal antibodies

A variety of cells originating from different species, including man, can be infected with Borna disease (BD) virus. Two different virus infection-specific antigens with molecular weights of 24 kD and 35/38 kD (double band) could be demonstrated using antigen preparations from persistently infected cells and rat brains, and polyvalent antisera from naturally and experimentally infected animals. Three different monoclonal antibodies were selected. One was specific for the 24-kD protein and two others reacted with the 35/38-kD antigen. The 24- and 35/38-kD antigens could be monitored concomitantly with the appearance of newly synthesized infectious virus. Both antigens could be detected at the same time during the infection cycle, and showed identical distribution in the cell. Their relationship to one another and their possible function is discussed.     

An enzyme-linked immunosorbent assay for the detection of antibodies to Marek's disease virus

A reproducible enzyme-linked immunosorbent assay (ELISA) using Marek's disease virus (MDV)-infected cells for the detection of antibodies to MDV is described. The optimum number of MDV-infected chicken embryo fibroblasts (CEF) was 5 X 10(4)/well, and test sera were positive at 1:400 dilutions. Compared with a purified virus preparation, MDV-infected CEF produced high specific and low nonspecific reactivities. Wells coated with whole cells could be stored at 4 C or -20 C for at least 3 months without loss of reactivity. With antibody-negative sera, the cutoff absorbency was 0.20 units. The ELISA was 20-to-40-fold more sensitive than indirect immunofluorescence. Homologous combinations of antisera in wells coated with CEF infected with different MDV serotypes were more reactive at higher dilutions than were heterologous combinations. The procedure described is specific and suitable for large-scale screening of both chicken and monoclonal antibodies against MDV.     

禽流感病毒单克隆抗体的制备及其抗蛋白抗原的分析

以纯化的H9N2亚型禽流感病毒为抗原,免疫BALB/c小鼠,细胞融合后,经间接ELISA和血凝抑制试验(HI)筛选,获得了8株能稳定分泌抗禽流感病毒单克隆抗体的杂交瘤细胞株。特异性试验证明,8株杂交瘤细胞株诱生小鼠腹水的特异ELISA抗体效价可达1∶3.2×103~1∶5.1×106,其中2株HI效价达212。8株单抗与H5亚型血凝素分型抗原不发生血凝,与减蛋综合征(EDS-76)病毒、传染性支气管炎病毒(IBV)、新城疫病毒(NDV)均不反应。亚类鉴定证实,除1C7单抗为IgG2b外,其他7株均为IgG1亚类。Westernblotting试验分析初步表明,8株单抗至少针对纯化病毒粒子3种不同的蛋白抗原,其中3株针对核蛋白(NP),2株针对基质蛋白M1,2株针对血凝素HA/HA1。对感染细胞的Western blotting分析结果与纯化病毒结果基本一致,其中1株未明显沉淀纯化病毒粒子蛋白的单抗可以与感染细胞的M2蛋白多肽反应。     

Stimulation by heterotypic antigens of foot-and-mouth disease virus antibodies in vaccinated cattle

Immunisation of cattle with foot-and-mouth disease virus failed to raise a level of antibody that provides protection against heterotypic challenge. Further the 12S substructure, produced from the 146S particle, was ineffective in providing protection against challenge by homotypic virus. These findings suggest considerable antigenic differences in the virus serotypes and between the virus and its substructure. Inoculation of homologous 12S and heterologous 1246S and 12S antigens into vaccinated cattle, however, revealed antigenic relationship between different serotypes, and between serotypes and their substructures.     

Identification and characterization of fowlpox virus strains using monoclonal antibodies.

The use of 2 monoclonal antibodies (MAbs), P1D9 and P2D4, which recognize different fowlpox virus (FPV) antigens, for the identification and characterization of FPV strains was evaluated. Initially, the MAbs were used in conjunction with a dot blot assay that enabled FPV to be differentiated from the avian herpesvirus, infectious laryngotracheitis virus. Confirmation of the specificity of these MAbs was provided by the demonstration that only FPV antigens were recognized by a combination of both antibodies when used for immunoblotting proteins contained in various avipoxviruses. Later, an antigenic characterization of 11 FPV field isolates, 6 FPV vaccine strains, and 3 pigeonpox virus vaccines was performed by Western blotting with the individual MAbs. Whereas MAb P2D4 consistently recognized a protein with an apparent molecular weight of 60 kD, there was variability in the size of the antigen that was immunoreactive with the other MAb. For example, MAb P1D9 recognized an antigen of apparent molecular weight of 46 kD in all vaccine strains except 2 of FPV origin. In these exceptions, either only a 39-kD or both a 42- and 46-kD protein were immunoreactive. As for the field isolates, a 39-kD antigen was recognized in 8 of them, whereas a 42-kD antigen was detected in the remaining 3. Therefore, the more extensive immunoblotting technique may facilitate FPV strain differentiation, whereas routine diagnosis of fowlpox could be accomplished by using the MAb-based dot blot assay.     

Identification of highly specific and cross-reactive antigens of Leishmania species by antibodies from Leishmania (Leishmania) chagasi naturally infected dogs

The Leishmania species present a genetic homology that ranges from 69 to 90%. Because of this homology, heterologous antigens have been used in the immunodiagnosis and vaccine development against Leishmania infections. In the current work, we describe the identification of species-specific and cross-reactive antigens among several New World Leishmania species, using symptomatic and asymptomatic naturally Leishmania chagasi-infected dog sera. Soluble antigens from five strains of New World Leishmania were separated by electrophoresis in SDS-PAGE and immunoblotted. Different proteins were uniquely recognized in the L. chagasi panel by either symptomatic or asymptomatic dog sera suggesting their use as markers for the progression of disease and diagnosis of the initial (sub-clinical) phase of the infection. Cross-reactive antigens were identified using heterologous antigenic panels (L. amazonensis strains PH8 and BH6, L. guyanensis and L. braziliensis). L. guyanensis panel showed the highest cross-reactivity against L. chagasi specific antibodies, suggesting that proteins from this extract might be suitable for the diagnosis of visceral canine leishmaniasis. Interestingly, the 51 and 97 kDa proteins of Leishmania were widely recognized (77.8% to 100%) among all antigenic panels tested, supporting their potential use for immunodiagnosis. Finally, we identified several leishmanial antigens that might be useful for routine diagnosis and seroepidemiological studies of the visceral canine leishmaniasis.     

超强马立克氏病病毒的鉴定及病毒Meq基因序列的比较

对1例感染超强马立克氏病病毒的病例进行确诊,对感染病毒的Meq基因进行比较分析。采用病理解剖、PCR检测、病毒分离、动物试验和Meq基因序列分析,对感染病毒进行研究。病理解剖结果为病死鸡的肝脏、脾脏、腺胃、肌胃、十二指肠表现为肿瘤病变。PCR检测结果为病死鸡的组织病料感染马立克氏病病毒。病毒分离和动物试验结果证明该感染病毒是1株马立克氏病超强毒株,该病毒可以引起免疫过CVI988疫苗的鸡发病。Meq基因序列分析表明该病毒与7株马立克氏病病毒参考毒株的同源性为98.8%~99.6%,该病毒在Meq的第115、119和176位氨基酸突变同国内流行株,该检测病毒在Meq的第217位氨基酸突变同超超强马立克氏病毒株。结果表明,通过病理解剖、PCR检测、基因序列分析、病毒分离和动物试验,确诊病鸡感染超强马立克氏病病毒。