Development and characterization of monoclonal antibodies to chicken interleukin-15

The chicken IL-15 gene was recently cloned and shown to encode a polypeptide with T cell growth factor activity similar to IL-2. To further characterize the chemical and biological properties of chicken IL-15, we generated a panel of monoclonal antibodies against bacterially expressed protein and characterized their binding specificities. All antibodies were reactive by ELISA with recombinant IL-15, but not IL-2, and identified a 15kDa recombinant chicken IL-15 by Western blot analysis. Two antibodies inhibited IL-15-induced proliferation of splenic lymphoblast cells. These monoclonal antibodies will be useful for further structural and immunological studies of chicken IL-15.     

Development and functional characterization of monoclonal antibodies recognizing chicken lymphocytes with natural killer cell activity.

We have developed two monoclonal antibodies which detect cell surface antigens present on chicken lymphocytes mediating natural killer (NK) cell activity against the avian tumor cell target. The monoclonal antibodies, K-14 and K-108, stained 17 and 6% of splenic lymphocytes, and 11 and 14% of peripheral blood lymphocytes (PBL), respectively, and fewer than 5% of thymic and bursal lymphocytes. Neither of these monoclonal antibodies stained adherent macrophages or the MC29-virus transformed monocytic cell line. Both monoclonal antibodies significantly inhibited NK cell activity in a standard 4 h 51Cr-release cytotoxicity assay using the LSCC-RP9 tumor cell line as target cells at an effector to target ratio of 50:1. Pretreatment of splenocytes with either monoclonal antibody in the presence of rabbit complement (C) resulted in a significant reduction in NK cell activity. However, the monoclonal antibody K-1 which detects normal chicken macrophages did not interfere with NK cell activity. The monoclonal antibody K-108 significantly blocked Fc receptor-mediated rosette formation of sheep red blood cells coated with IgG antibodies (EA) by 56% while the monoclonal antibody K-14 did not show a significant blocking. These results indicate that the monoclonal antibodies K-108 and K-14 identify different epitopes present on the surface of chicken splenic lymphocytes which mediate spontaneous NK cytotoxicity.     

Development and characterization of mouse monoclonal antibodies reactive with chicken CD80

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD80 (chCD80). A recombinant plasmid containing a chCD80/horse IgG4 fusion gene was constructed and expressed in CHO cells to produce recombinant chCD80/IgG4 protein. Chicken CD80 was purified from the chCD80/IgG4 fusion protein following enterokinase digestion, and used to immunize BALB/c mice, resulting in 158 hybridomas that produced mAbs against chCD80. Three mAbs with high binding specificity for recombinant chCD80/IgG4-transfected CHO cells were identified by flow cytometry, and one of these (#112) was selected for further characterization. Immunoprecipitation of CD80/IgG4-CHO cell extract, or lipopolysaccharide (LPS)-treated monocytes identified 35.0 kDa proteins. Immunohistochemical analysis revealed chCD80-expressing cells exclusively in the bursal follicles at the outer portion of the cortex, and throughout the red pulp and the outer boundary of the white pulp in the spleen. By immunofluorescence microscopy, chCD80 was observed on intestinal dendritic cells. LPS treatment of bursa or spleen monocytes for 24 or 48 h increased chCD80 expression. Finally, addition of chCD80 mAb to Con A-stimulated spleen cells inhibited the expression of major histocompatibility complex class II antigens and IL-2-driven proliferation of lymphoblast cells. In summary, these chCD80 mAbs will serve as valuable immunological reagents for basic and applied poultry immunology research.     

Development and characterization of mouse monoclonal antibodies reactive with chicken CD83

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD83 (chCD83), a membrane-bound glycoprotein belonging to the immunoglobulin superfamily that is primarily expressed on mature dendritic cells (DCs). A recombinant chCD83/IgG4 fusion protein containing the extracellular region of chCD83 was expressed in Chinese Hamster Ovary (CHO) cells and isolated from the spent cell culture medium by protein G affinity chromatography. The extracellular region of the chCD83 protein was purified and used to immunize mice. A cell fusion was performed, from which 342 hybridomas were screened for mAbs to chCD83. Two mAbs, chCD83-159 and chCD83-227, stained the greatest percentage of chCD83-transfected CHO cells and were selected for further characterization. By flow cytometry, both mAbs reacted with a chicken macrophage cell line, HD11. Both mAbs also recognized a single 53 kDa protein on Western blots of lysates from lipopolysaccharide-stimulated spleen mononuclear cells or unstimulated HD11 cells. Immunostaining of chicken secondary lymphoid organs identified chCD83(+) cells with morphologic and subtissue localization properties comparable to mammalian DCs. In vitro stimulation of spleen mononuclear cells with concanavalin A (Con A) decreased the percentage of chCD83(+) cells compared with cells treated with medium alone. Interestingly, spleen cells treated with Con A in the presence of chCD83-227 mAb exhibited decreased percentage of MHCII(+) cells compared with cells treated with an isotype-matched negative control mAb. These chCD83 mAbs may be useful for future investigations of chicken immune cell maturation and mechanisms of action.     

Production and preliminary characterization of monoclonal antibodies to chicken anemia agent

Mice were immunized with partially purified preparations of the Cux-1 isolate of chicken anemia agent (CAA), and their splenocytes were fused with NSO myeloma cells. Three patterns of staining of CAA-infected cells were recognized when the resulting hybridomas were screened by indirect immunofluorescence (IIF). Hybridomas representative of each staining pattern were cloned, and the monoclonal antibodies (MAbs) were characterized. Type 1 staining was indistinguishable from that produced by polyclonal chicken antisera to CAA. Type 2 staining was confined to large nuclear inclusions. Type 3 staining was predominantly nuclear and granular, and differed from type 1 in being more intense and occurring in a higher proportion of nuclei. Three MAbs producing type 1 staining were predominantly Cux-1-specific by IIF; they also reacted to lower titers with the Gifu-1 isolate but not at all with three other CAA isolates. These MAbs had very slight neutralizing activity against Cux-1. Another MAb giving type 1 staining reacted with all CAA isolates tested to high titers in IIF and neutralization tests. MAbs with type 2 and type 3 staining reacted by IIF with all CAA isolates tested but possessed no neutralizing activity. The availability of MABs to CAA should facilitate development of diagnostic tests for the virus.     

Characterization and use of monoclonal antibodies to identify Haemophilus paragallinarum serovars

Two serovar-specific monoclonal antibodies (MAbs) to Haemophilus paragallinarum serovars A/1 and C/2 strains, respectively, were developed and characterized by hemagglutination-inhibition (HI) and dot-blotting tests using representative H. paragallinarum serovars A/1, B, and C/2 strains. In both the HI and dot-blotting tests, one MAb (E5C12D10), raised against strain 221, serovar A/1, reacted only with serovar A/1 strains, while the other MAb (F2E6), raised against strain S1 of serovar C/2, reacted with only serovar C/2 strains examined. In both tests, the two MAbs did not react with two serovar B strains. These results indicated that the two MAbs recognize serovar-specific hemagglutinating (HA) antigens of H. paragallinarum serovars A/1 and C/2 strains, respectively, and that a dot-blotting test using these MAbs is a practical alternative to the HI test for serotyping H. paragallinarum. Strains 0222 and Spross of serovar B, which did not react with these two MAbs, were found to possess serovar-specific HA antigen in cross-HI tests.     

H5亚型禽流感单克隆抗体的研制及应用

以禽流感H5亚型病毒分别免疫BABL／C小鼠，取其脾细胞与SP2／0骨髓瘤细胞融合，用血凝抑制试验(HI)和间接ELISA检测细胞上清液，结果获得了3株抗禽流感H5亚型病毒特异性单克隆抗体，分别命名为186，1D4，7D1；其中1D4株为抗禽流感H5亚型病毒血凝素特异性单克隆抗体细胞株．186株和7D1株为针对禽流感病毒NP蛋白的单克隆抗体细胞株。经3次亚克隆后，100％杂交瘤细胞保持了分泌抗禽流感病毒抗体的能力。这些单克隆抗体小鼠腹水ELISA效价为10^5，HI效价为2^11-12。研究结果表明，所有这些单克隆抗体仅与相应的禽流感病毒株发生特异性反应，而不与鸡新城疫病毒、产蛋下降综合征病毒、鹅副粘病毒等反应。所有这些单抗在禽流感诊断中将发挥重要作用。     

Development and characterization of monoclonal antibodies to spring viraemia of carp virus

Five monoclonal antibodies (mAbs) against spring viraemia of carp (SVCV0504, isolated from common carp in China) were produced from mice immunized with purified virus preparations. The virion of SVCV contains five structural proteins, representing the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). Western blotting analysis revealed that three mAbs (1H5, 1E10, and 1H7) recognized specifically to a single protein of 47kDa (N), the mAb 3G4 reacted with two SVCV0504 proteins of 69kDa (G) and 47kDa (N), while the mAb 1A9 reacted with three SVCV0504 proteins of 69kDa (G), 50kDa (P), and 47kDa (N). By indirect ELISA, two mAbs (1H5 and 1H7) showed cross-reactivity with pike fry rhabdovirus (PFRV), but no cross-reactions with the Siniperca chuatsi rhabdovirus (SCRV), Scophthalmus maximus rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (PoRV) were demonstrated with the five mAbs. Indirect immunofluorescence showed intense fluorescence in the cytoplasm of the SVCV0504-infected epithelioma papulosum cyprini (EPC) cells in areas corresponding to the location of granular structures. The sucrose gradient-purified SVCV0504 particles could be detected successfully by these mAbs using immunodot blotting. mAb 1A9 could completely neutralize 100 TCID(50) (50% tissue culture infective dose) of SVCV0504 at a dilution of 1:8. This is the first report of development of the neutralizing mAbs against SVCV. The mAb 1A9 was analyzed further and could be used to successfully detect viral antigens in the infected-EPC cell cultures or in cryosections from experimentally infected crucian carp (Carassius auratus) by immunohistochemistry assay. Furthermore, a flow cytometry procedure for the detection and quantification of cytoplasmic SVCV0504 in cell cultures was developed with mAb 1A9. At 28h after inoculation with the virus (0.01PFU/cell), 10.12% of infected cells could be distinguished from the uninfected cells. These mAbs will be useful in diagnostic test development and pathogenesis studies for fish rhabdovirus.     

Development and characterization of monoclonal antibodies to subgroup A avian leukosis virus

Avian leukosis virus subgroup A (ALV‐A) is a retrovirus which infects egg‐type chickens and is the main pathogen of lymphoid leukosis (LL) and myeloid leukosis (ML). In order to greatly enhance the diagnosis and treatment of clinical avian leukemia, two monoclonal antibodies (MAbs) to ALV‐A were developed by fusion between SP2/0 and spleen cells from mice immunized with expressed ALV‐A env‐gp85 protein. Using immunofluorescence assay (IFA), two MAbs reacted with ALV‐A, but not with subgroups B and J of ALV. Western blot tests showed that molecular weight of ALV‐A envelope glycoprotein recognized by MAbs was about 53 kD. Isotyping test revealed that two MAbs (A5C1 and A4C8) were IgG1 isotypes. These MAbs can be used for diagnosis and epidemiology of ALV‐A.     

SMD单克隆抗体的制备及测定方法的建立

用戊二醛作为偶联剂 ,将磺胺对甲氧嘧啶 (SMD )与牛血清白蛋白 (BSA )或卵清蛋白 (OVA)偶联形成完全抗原 ,经紫外分光光度计扫描鉴定。以人工抗原免疫BALB/c小鼠 ,取脾细胞与骨髓瘤细胞 (SP2 /0 -Ag1 4)融合 ,用间接ELISA联合竞争ELISA法筛选出产生针对SMD抗体的杂交瘤细胞。经克隆 ,得到 3株特异性好的阳性杂交瘤细胞 ,注入小鼠腹腔产生腹水。建立了测定SMD的ELISA法 ,其检测下限小于5ng/mL。     

Production and characterization of monoclonal antibodies reactive with the chicken interleukin-15 receptor alpha chain

We recently cloned the genes encoding chicken IL-15 and IL-15 receptor (R) alpha proteins. In this study, 12 monoclonal antibodies (mAbs) against recombinant chicken IL-15Ralpha were produced and characterized. By enzyme-linked immunosorbent assay (ELISA), all mAbs showed binding specificity for IL-15Ralpha, but not IL-2 or interferon-gamma, and identified a 25.0kDa protein by immunoblot analysis. Flow cytometric analysis revealed negligible expression of IL-15Ralpha on non-activated lymphocytes from the spleen, thymus or bursa, low but detectable expression on macrophages and high expression on concanavalin A-activated spleen lymphoblasts. Established chicken T cell (RP13) and macrophage (HD11) cell lines expressed substantially higher levels of IL-15Ralpha compared with a B cell line (RP9). Two mAbs inhibited IL-15 dependent proliferation of T cells suggesting that the tertiary structure of the protein domain of native IL-15Ralpha that binds to IL-15 is preserved in the recombinant receptor molecule. These mAbs will be useful reagents for further in vitro and in vivo studies of the biological functions of chicken IL-15 and its receptor.     

Production and characterization of monoclonal antibodies that react to the nucleocapsid protein of avian metapneumovirus subtype C

Monoclonal antibodies (MAbs) were prepared against avian metapneumovirus (aMPV) subtype C (aMPV/ Minnesota/turkey/1a/97). Six MAbs were selected based on enzyme-linked immunosorbent assay activities and characterized by isotyping, neutralization test, western blot analysis, and immunohistochemistry assay. The results showed that three MAbs (3E, 9D, and 12C) belonged to the IgG1 subclass, whereas the other three (5D, 8E, and 16E) were of the IgG2a subclass. None of the six MAbs neutralized aMPV infectivity at a detectable level, but all reacted with both denatured and nondenatured forms of the nucleocapsid (N) protein of aMPV, suggesting that these MAbs may recognize structurally independent epitopes of the N protein. These MAbs provide new tools and methods for investigating aMPV infection and pathogenesis, as well as diagnosis of aMPV disease.     

Oxidative and nitrosative responses of the chicken macrophage cell line MQ-NCSU to experimental Salmonella infection

Phagocytes limit replication or kill ingested organisms by producing toxic reactive oxygen and nitrogen species via NADPH oxidase and inducible nitric oxide synthase (iNOS). The present experiments were to investigate the production and the possible roles of superoxide, hydrogen peroxide (H2O2) and nitric oxide (NO) in the MQ-NCSU chicken macrophage cell line infected with Salmonella in vitro. After infection, intracellular Salmonella viable counts remained constant until 24 h post infection (PI) and started to decline from 48 h PI. Infection of cells with S. Typhimurium, S. Enteritidis and S. Gallinarum, as well as exposure to S. Enteritidis LPS induced low, but significant concentrations of superoxide 1 to 2 h PI, as determined by reduction of ferricytochrome c. There was no difference in superoxide production in infected cells and control cells after 4 h. Increased H2O2 was observed from cells infected with all the different Salmonella species between 2 and 3 h of infection. Nitrite was always greater in infected cells compared to uninfected cells at all times. However, Salmonella was not completely eliminated from the cells though these cells are capable of eliciting a noticeable oxidative burst response and great nitrosative responses, indicating that a strong oxidative burst (and other mechanism/s) is essential for the elimination of intracellular Salmonella.     

Development and characterization of monoclonal antibodies to first-generation merozoites of Eimeria bovis.

Merozoites of Eimeria bovis were harvested from bovine monocyte cell cultures and used to immunize BALB/C mice. Spleens from immunized mice were removed and the cells fused with mouse myeloma cells. Supernates from resulting hybridoma cell lines were examined for antibodies to first-generation E. bovis merozoites using an indirect immunofluorescent antibody (IFA) assay. Three positive cell lines were identified and cloned by limiting dilution. All three cell lines produced immunoglobulins of the IgG1 isotype that recognized antigens in the anterior half to two-thirds of the merozoites. Specificity of the monoclonal antibodies was examined with the IFA assay against sporozoites of E. bovis, sporozoites and merozoites of Eimeria papillata from mice and Eimeria tenella from chickens, sporozoites of Isospora suis from pigs, and tachyzoites of Toxoplasma gondii and Neospora caninum from cell cultures. Monoclonal antibodies from the three clones reacted with the anterior end of E. bovis sporozoites, but did not react with the other parasites examined. None of the monoclonal antibodies reacted with merozoite antigens in immunoblots.     

Effect of neutralizing monoclonal antibodies on Hantaan virus infection of the macrophage P388D1 cell line.

The effect of neutralizing monoclonal antibodies (N MAbs) on Hantaan virus infection of macrophages was investigated using P388D1 cells, a murine macrophage cell line. MAbs to the G1 protein (G1b) and the G2 protein (G2a and G2c) neutralized viral infectivity in P388D1 cells. N MAbs to G1b showed much higher neutralizing potency than those to G2a and G2c. With each N MAbs, two distinct effects were observed: neutralization of viral infectivity occurring at high concentrations and enhancement of that at low concentrations. Non-neutralizing MAbs, on the other hand, showed only enhancement of viral infectivity even at high concentrations without any inhibitory effects. The Fab fragments of N MAbs showed neither neutralizing nor enhancing activities. However, when the virus-Fab complexes were reacted with the anti-Fab antibodies, both neutralization and enhancement of viral infectivity were restored depending on the dose of Fab fragments. These results indicate that Hantaan virus infection of P388D1 cells is mediated by the Fc portion of the antibodies and neutralization is dependent on the concentration of N antibodies bound bivalently to the neutralization site on the virion.     

Development and characterization of monoclonal antibodies to subgroup J avian leukosis virus.

In an attempt to develop a specific diagnostic test for avian leukosis virus (ALV) subgroup J (ALV-J) strain Hc1, four monoclonal antibodies (MAbs), JE9, G2, 145, and J47, were generated that are specific for ALV-J envelope glycoprotein, gp85. Polymerase chain reaction (PCR) was used to amplify genomic pro-viral DNA of Avian Disease and Oncology Laboratory (ADOL)-Hc1 and ADOL-4817 envelope genes. Both open reading frames encoding glycoproteins gp85 and gp37 were cloned into baculoviruses. Abundant expression of gp85 and gp37 was detected in the recombinant viruses with specific antibody to Hc1 strain of the ALV-J. The expressed proteins were used for immunization of mice to produce hybridoma cell lines secreting MAbs specific to ALV-J envelope protein. A panel of MAbs was generated by fusing NS1 myeloma cells and spleen cells from mice immunized with the recombinant baculoviruses. With the use of an immunofluorescence assay, three MAbs (JE9, G2, 145) reacted with ALV-J but not with subgroups A, B, C, D, or E of ALV. MAb J47 reacted with all exogenous subgroups of ALV including A, B, C, D, and J but not with endogenous subgroup E viruses. Western blot analysis was performed with all four MAbs against recombinant baculovirus and Hc1-infected chicken embryo fibroblast (CEF) lysates. A major band with a molecular weight about 90 kD corresponding to the size of ALV-J envelope was consistently obtained. With these MAbs, we detected the Hc1 antigen in CEFs infected with several ALV-J viruses isolated in the United States and also in tissue sections from chickens infected with Hc1 strain of ALV-J. These MAbs will be useful reagents for the diagnosis of ALV-J infection because they recognize a common antigenic epitope in six isolates tested thus far.     

抗鸡IBDV Gt株单克隆抗体制备及抗原表位初步分析

采用不连续蔗糖密度梯度离心法纯化的鸡传染性法氏囊病病毒Gt株免疫BALB/C小鼠,利用淋巴细胞杂交瘤技术,取其脾细胞与SP2/0骨髓瘤细胞进行融合,经过三次筛选克隆,获得具有分泌抗IBDV抗体的7D_4、5D_2两株杂交瘤细胞系,并对7D_4及5D_2株进行了特异性、稳定性等方面的鉴定。试验表明,两株分泌的抗体能够特异地与IBDV VP2蛋白反应。7D_4株及5D_2株杂交瘤细胞上清ELISA效价分别为1：250、1：50,腹水ELISA效价为6．4×10~6、5×10~3；并运用7D_4、5D_2介导的相加ELISA进行抗原表位的初步定位,这两株单抗针对不同的VP2抗原表位。     

Isolation of monoclonal antibodies that inhibit the binding of infectious bursal disease virus to LSCC-BK3 cells

Three hybridoma cell lines producing monoclonal antibodies (MAbs) against LSCC-BK3 cells which are susceptible to infectious bursal disease virus (IBDV) infection were produced and characterized. The MAbs, designated T7, Q11 and Q13, inhibited the attachment of IBDV to LSCC-BK3 cells. Furthermore, these MAbs bound to LSCC-BK3 but not to nonpermissive cells in flow cytometry. MAb T7 detected a 110-kDa membrane protein of LSCC-BK3 cells, whereas Q11 and Q13 reacted with membrane proteins of molecular weights 58-, 85-, 90- and 110-kDa. These observations imply that the 110-kDa protein recognized by all the MAbs is associated with IBDV binding. The MAbs established in this study are useful for studying the interaction between IBDV and its target cell.     

流行性乙型脑炎病毒E蛋白单克隆抗体的制备与鉴定

流行性乙型脑炎病毒(JEV)是一种严重危害人畜健康的虫媒病毒。表面囊膜蛋白(E蛋白)是该病毒的主要结构蛋白。本研究利用原核表达的乙型脑炎病毒SA14-14-2株结构蛋白E蛋白作为免疫原,免疫6周龄BALB/c小鼠,采用淋巴细胞杂交瘤技术进行融合,经有限稀释法获得1株特异性针对JEV E蛋白的杂交瘤细胞,命名为5D4,经测定5D4单抗亚类属于IgG2a,轻链为κ链。经Western blot证实所得杂交瘤细胞分泌的抗体可特异地与JEV E蛋白结合。结果表明,所制备的抗JEV E蛋白的单抗对病原检测具有很高的特异性,为JEV准确快速的病原诊断和JEV感染相关的基础性研究提供了物质基础。