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.     

Development and characterization of a monoclonal antibody specific for bovine CD209

Dendritic cells (DC) play a central role in tailoring the immune response to pathogens. Effector activity is mediated through pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns (PAMPS). C-type lectin receptors (CLR) comprise a group of PRRs that recognize a broad range of pathogens. CD209 (DC-specific ICAM3-grabbing non-integrin, DC-SIGN) is a CLR expressed on DC that plays a critical role on DC function and pathogen recognition. It facilitates DC migration to peripheral tissues and local lymph nodes and mediates T cell activation by binding ICAM-2 (CD102) and ICAM-3 (CD50). The absence of monoclonal antibody (mAb) to bovine CD209 has limited the ability to characterize the phenotype and function of DC in cattle. To address this issue we developed and used a mAb to CD209 to characterize the phenotype of CD209 expressing cells in bovine blood using flow cytometry. Initial analysis has revealed the CD209 positive population in blood is comprised of multiple phenotypically defined subsets.     

Production and characterization of monoclonal antibodies detecting chicken interleukin-2 and the development of an antigen capture enzyme-linked immunosorbent assay

Eleven monoclonal antibodies (mAbs) which are specific for chicken interleukin-2 (chIL-2) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting and neutralizing assays. These mAbs were used to develop a mAb-based antigen capture ELISA specific for chicken IL-2 detection. Anti-IL-2 mAbs bound specifically to E. coli-derived rchIL-2 in ELISA and identified a 16 kDa IL-2 polypeptide band in Western blot. Several mAbs were shown to neutralize the biological activities of both rchIL-2 and native chicken IL-2 as measured by concanavalin A (ConA)-induced lymphocyte proliferation assay, IL-2 bioassay, and natural killer cell assay. Among the neutralizing mAbs, the mAb chIL-2/11 was most potent in neutralizing IL-2 activity. To develop a sensitive ELISA for the detection of chicken IL-2, an antigen capture ELISA was developed using the mAb chIL-2/16 as the antigen capture antibody and rabbit anti-IL-2 peptide antibody as the detection antibody. Using the mAb-based antigen capture ELISA, significant correlation between the level of IL-2 detected in bioassays and in ELISA was observed. These results showed that the mAb-based antigen capture ELISA is less time-consuming and more reliable compared to a conventional IL-2 bioassay for chicken IL-2. These neutralizing mAbs will facilitate basic immunobiological studies of the role of IL-2 in normal and disease states in chickens.     

Analyses of monoclonal antibodies reacting with porcine CD5: results from the Second International Swine CD Workshop

Among the 57 monoclonal antibodies analyzed within the T-cell group, three mAbs fell within cluster T13 including the CD5a standard b53b7 (No. 174). The two new mAbs 1H6/8 (No. 058) and BB6-9G12 (No. 166) both precipitated 55 and 60 kDa proteins that were of similar molecular weights as the standard. Staining patterns on the various cell types were similar. Both new antibodies inhibited the binding of the CD5a reference mAbs b53b7 to peripheral lymphocytes. These mAbs, therefore both react with the CD5a epitope bringing the number of anti-porcine CD5 mAbs to eight, all of which appear to recognize the same epitope.     

鸡白细胞介素2分子单克隆抗体的研制

以含有鸡白细胞介素2（ChlL-2）基因的重组真核表达质粒pcDNA3．1-ChlL-2免疫6周龄BALB／c小鼠,末次加强免疫后取其脾细胞与骨髓瘤细胞Sp2／0-Ag-14融合,应用间接ELISA筛选阳性克隆。共获得2株特异性分泌抗鸡IL-2分子的杂交瘤细胞株,分别命名为1H10和1E6,其腹水ELISA效价分别为1：6400和1：3200,亚类鉴定结果均为IgM。重组质粒pcDNA3．1-ChlL-2转染COS-7细胞,以上述单克隆抗体（mAb）检测表达产物,结果表明,2株mAb均能与表达产物反应;Western blot分析结果显示,2株mAb均与原核表达鸡IL-2蛋白发生特异性反应。本研究所制备的抗鸡IL-2分子mAb可为建立简单、快速的鸡IL-2检测方法提供可能,并为鸡IL-2生物学特性和禽类细胞免疫机理的研究提供材料。     

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.     

猪圆环病毒2型感染对猪CD80和CD86 mRNA表达的影响

通过构建猪共刺激分子CD80和CD86的缺失cDNA竞争分子,用竞争PCR技术定量检测了猪圆环病毒2型(PCV2)感染后猪肺泡巨噬细胞(PAM)中CD80和CD86的mRNA水平,分析了PCV2感染对猪共刺激分子CD80和CD86的mRNA表达的影响.结果显示,PCV2感染后,PAM中CD80和CD86的mRNA水平变化趋势一致,只是CD86的升降幅度更大.在PCV2感染后3d,CD80与CD86的mRNA水平下降至最低,随后迅速上升,至14d达到高峰,尔后快速下降,21d及以后恢复正常.本研究结果表明,PCV2初期可明显抑制猪共刺激分子CD80和CD86的基因转录.     

Analyses of monoclonal antibodies reacting with porcine CD3: results from the Second International Swine CD Workshop

Among the 57 monoclonal antibodies (mAb) analyzed within the T-cell group from the Second Swine CD Workshop, six mAb fell within clusters T10 and T11 (No. 088, STH164; No. 148, FY1A3; No. 149, FY2C1; No. 150, FY1H2; No. 151, FY2A11; No. 169, BB23-8E6). The mAb within these two groups gave a similar appearance on flow cytometry and stained all peripheral blood T-cells as defined by CD4 and wCD8 staining. All six mAb precipitated a 24 kDa protein. On the basis of inhibition analyses performed as part of the workshop and from published data, the mAb define at least three epitopes. There is only minimal stimulation of resting peripheral lymphocytes, but four of the mAb produce strong stimulation in the presence of PMA. With the exception of STH164, all have been shown to react with CD3-transfected COS cells. The new mAb, therefore, react with three epitopes on porcine CD3 designated CD3a (BB23-8E6, FY2A11), CD3b (FY1A3, FY2C1), and CD3c (FY1H2). mAb STH164 appears to be reactive with another epitope, however, since its reactivity with CD3 has not been confirmed it is designated as wCD3.     

Development and characterization of mouse monoclonal antibodies to eight human complement components: Analysis of reactivity with orthologs of nine mammalian genera

To study complement function in mammalian leishmanioses, we developed mouse monoclonal antibodies to the human complement system components C1q, C4, factor D, factor H, factor B, properdin, C5 and C9. Antibody specificity was determined by indirect and capture ELISA and by Western blot. In flow cytometry analysis, seven antibodies recognized the cognate component on human serum-opsonized Leishmania promastigotes. Antibody reactivity was screened against promastigotes opsonized with sera of nine mammalian genera: pig, guinea pig, goat, rabbit, cat, dog, hamster, jird and rat. No antibody recognized jird epitopes on promastigotes. Anti-C4, -properdin, and -C5b reacted with the orthologous protein of all other mammals tested except cat (anti-properdin) and hamster (anti-C5b); anti-C9 only recognized the rabbit ortholog, and anti-C1q, -factor B and -factor H did not react with any of the nine orthologs. Such interspecies crossreactive antibodies can be valuable tools for analysis of mammalian complement function in infectious diseases.     

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.     

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.     

Workshop studies with monoclonal antibodies identifying a novel porcine differentiation antigen, SWC9

Two monoclonal antibodies (mAb) within cluster M4 of the myeloid section of the Second International Swine CD Workshop, C4 (No. 144) and PM18-7 (No. 192), showed reactivity with thymocytes and among cells of myelomonocytic origin with mature macrophages but not with monocytes and granulocytes. Both mAb recognize a protein showing two bands of 205 kDa and 130 kDa under both reducing and non-reducing conditions. Although epitope mapping with these mAb could not be performed, this cluster received the SWC9 designation.     

Definition of the specificity of monoclonal antibodies against porcine CD45 and CD45R: report from the CD45/CD45R and CD44 subgroup of the Second International Swine CD Workshop

Swine cell binding analyses of a set of 48 monoclonal antibodies (mAbs), including eleven standards, assigned to the CD44 and CD45 subset group of the Second International Swine CD Workshop yielded 13 clusters. Although none of these corresponded to CD44, seven mAbs formed a cluster which was identified as being specific for restricted epitopes of CD45 (CD45R). In addition, a T-cell subset specific cluster comprised of four mAbs was also identified. Two mAbs (STH106 and SwNL 554.1) reacted exclusively with CD8 bright lymphocytes, the other two (2B11 and F01G9) with a subset of CD4 lymphocytes. The other 10 clusters were either specific for MHC-class I like molecules or overlapped with clusters identified by the adhesion molecule subgroup and are therefore just briefly discussed in this report. The specificity of all the mAbs in the CD45R cluster was verified by their ability to immunoprecipitate distinct proteins and to react with CHO cells expressing individual isoforms of CD45. Three CD45R mAbs (3a56, MIL5, −a2) did react with a 210 kDa isoform(s), while another three (STH267, FG2F9, 6E3/7) only recognized a 226 kDa isoform(s). The remaining one (MAC326) precipitated both a 210 and 226 kDa protein. The specificity of all the mAbs in the CD45R cluster, and of the CD45 common mAbs, was confirmed by their reactivity with CHO cells transfected with cDNAs encoding the extracellular and transmembrane portions of distinct CD45R isoforms. Those mAbs recognizing a 210 kDa protein reacted with CHO cells expressing the CD45RC isoform, while those capable of precipitating a 226 kDa, but not the 210 kDa, polypeptide recognized CHO cells expressing either the CD45RAC and the relatively rare CD45RA isoform. MAC326 was unique in its inability to react with CHO cells engineered to produce the CD45RC and CD45RAC isoforms. Thus, three mAbs (6E3/7, STH267, and FG2F9) appear to be specific for an epitope(s) encoded by the A exon, while one (MAC326) recognizes a determinant encoded by the C exon. The remaining three mAbs (3a56, −a2, MIL5) are apparently specific for an epitope(s) which results from the fusion of the C exon to the invariant leader sequence and is destroyed by inclusion of the A exon. All three CD45 common mAbs, K252.1E4, MAC323 and 74.9.3, did react with the CHO cells lines expressing either the CD45RA, CD45RC, CD45RAC or CD45RO isoforms, but not with untransfected CHO cells. When the natural expression of CD45 isoforms was examined by reacting lymphocytes with CD45R mAbs, a high level expression of isoforms containing the A exon-generated domain was detected in all B cells while the majority of CD4⁺ T cells had undetectable or lower expression density of this protein than B cells. In contrast, the density of expression of the CD45 isoform(s) containing the C exon-generated domain ranged from undetectable to high in CD4⁺ T cells whereas the amounts were approximately ten-fold lower in B cells. Overall this panel of CD45 mAbs will be very useful in analyzing the maturation and differentiation of swine lymphoid cells subsets.     

鸡Mx蛋白单克隆抗体的制备及鉴定

Mx蛋白为GTP酶动态蛋白家族成员,已被证明具有抗禽流感病毒、Thogoto病毒和水泡性口膜炎病毒等的作用.为制备鸡Mx蛋白的单克隆抗体(MAb),本研究以原核表达并纯化的鸡Mx蛋白为免疫原,免疫BALB/c小鼠,分离脾细胞与SP2/0细胞融合,采用间接ELISA方法筛选阳性克隆,经3次细胞克隆纯化后获得4株稳定分泌抗鸡Mx蛋白抗体的杂交瘤细胞株.间接ELISA测定4株MAb纯化后腹水的效价介于1∶3.2×104～1∶2.5×105之间.Westem blot结果证实4株MAb均具有良好的反应性;间接免疫荧光试验表明其中一株MAb与真核表达的鸡Mx蛋白发生反应.本研究为鸡Mx蛋白功能及表达鸡Mx蛋白的转基因动物等研究奠定了基础.     

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.     

快速、特异检测李斯特菌单抗-夹心ELISA方法的建立及应用

本研究以高度特异的、针对单核细胞增生李斯特氏菌、无害李斯特氏菌、格氏李斯特氏菌共同表位的单抗ＬＪ１０Ａ为捕捉抗原抗体和酶标抗体,建立了快速检测该菌的单抗夹心ＥＬＩＳＡ方法。该方法对单核细胞增生李斯特菌的最小检出量为５×１０５个菌／ｍｌ。人工模拟样品至少可检出５个菌／１０ｇ样品,并在４８小时内报告结果。在实际应用中,我们以该法检测了２４０份肉样和８５０份奶样,并平行以改良ＭｃＢｒｉｄｅ琼脂分离细菌相比较,证实该方法的敏感性和特异性分别达到１００％和９６．９％。     

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.     

The risk of using monoclonal or polyclonal commercial antibodies: controversial results on porcine sperm CD44 receptor identification

Presence of the hyaluronan (hyaluronic acid, HA) receptor CD44 on spermatozoa has been difficult to pursue, mostly obeying to the use of different commercial mono‐ and/or polyclonal antibodies, often lacking proper controls. Here, we describe how the presence (Western blotting) and specific location (immunocytochemistry) of the CD44 receptor differs in ejaculated pig spermatozoa depending on the type of antibody and protocol used. While we were able to detect binding to spermatozoa and mark its presence in the sperm membrane, the use of blocking peptides clearly indicated that only the monoclonal antibody could confirm the specific presence and location of the CD44 receptor, whereas the polyclonal antibody was detecting multiple presumed CD44 isoforms or degraded proteins thus proving unspecific. These results call for strict protocols when attempting immunological determination of sperm membrane receptors.     

鸡白细胞介素18成熟蛋白的表达、纯化及其单克隆抗体的制备与特性鉴定

将鸡白细胞介素18成熟蛋白(mature chicken interleukin 18,mChIL-18)基因亚克隆至原核表达载体pET-28a(+),并在大肠杆菌中高效表达,采用Ni-NTA亲和层析方法纯化重组蛋白,免疫BALB/c小鼠,经融合、筛选制备特异性单克隆抗体(mAb),并通过间接ELISA、间接免疫荧光(IFA)和Western blot等方法对mAb做初步鉴定.结果表明,试验成功表达了mChIL-18蛋白,并获得2株持续且稳定分泌抗体的杂交瘤细胞(1G9、2E6),其腹水ELISA效价分别为1∶5.12×10 5和1∶3.2×104,亚类鉴定结果均为IgM.Western blot结果显示,2株单抗均能特异性识别原核表达的mChIL-18蛋白,IFA分析表明它们均能与真核表达的mChIL-18发生特异性反应,ELISA叠加试验证实1G9与2E6针对2个不同的抗原表位.本研究所获单抗可以用于建立鸡IL-18的检测方法,为进一步开展其生物学功能研究奠定了基础.