Production and characterization of monoclonal antibodies against porcine interleukin-4

Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. In the present study, recombinant thioredoxin (Trx)-porcine IL-4 (pIL-4) fusion protein was prepared by Escherichia coli (E. coli), and by using this protein as an immunogen, monoclonal antibodies (mAbs) against pIL-4 were produced to establish a basis for a research on immune responses in pigs. Six stable hybridoma cell lines were successfully established and specific binding of each mAb to recombinant pIL-4 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that the subclass of 5 out of 6 mAbs was IgG1 and the rest was IgG2b. Further, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to 4 different epitopes. The recombinant proteins and mAbs produced in this study will be useful tools for the assessment of porcine immune system.     

Development of a monoclonal blocking ELISA for the detection of antibodies against fowlpox virus

To provide a fast and easy method to detect antibodies against fowlpox virus (FWPV) particularly in high numbers of chicken sera we established a monoclonal blocking enzyme-linked immunosorbent assay (ELISA). We chose two different monoclonal antibodies (mAb), anti-FWPV 3D9/2B3 and anti-FWPV 8F3/2E11, which are both directed against the 39-kDa protein of FWPV strain HP-1. The blocking ELISA depends on the blocking of mAb binding to solid-phase antigen in the presence of positive serum. For an epidemiological study a total of 184 serum samples from Gambian chicken flocks were analysed against each of the mAbs. Four of the sera were shown to contain FWPV antibodies. These four sera showed a positive cut-off value of more than 50% inhibition exclusively in the test against the mAb anti-FWPV 8F3/2E11. This phenomenon can be explained by the binding of the mAbs to distinct epitopes on the same protein.     

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.     

Production and characterization of two serotype independent monoclonal antibodies against foot-and-mouth disease virus

Two foot-and-mouth disease virus (FMDV) monoclonal antibodies (mAbs) were produced from mice immunized with either FMDV serotype A, subunit (12S) or FMDV serotype O, whole virus (140S). Both mAbs (F1412SA and F21140SO) recognized all seven serotypes of FMDV in a double antibody sandwich (DAS) ELISA, suggesting that the binding epitopes of the two mAbs are conserved between serotypes. These mAbs are IgG1 isotype and contain kappa light chains. In order to define the mAb binding epitopes, the reactivity of these mAbs against trypsin-treated and denatured FMDV were examined using an indirect ELISA. The binding site of the mAb, F1412SA is trypsin sensitive and the epitope is linear. Both ELISA and Western blot results suggested that the polypeptide VP2 contributed to the immunodominant site. This mAb showed reactivity to VP2 peptide (DKKTEETTILEDRIL). The mAb, F21140SO, recognized an epitope which is trypsin resistant and discontinuous. This mAb binding to FMDV is dependent on conformational structures of intact viral (140S) or subunit (12S) particle, since it failed to recognize any viral protein in Western blot. This conformational and highly conserved epitope is the first identified epitope among all seven FMDV serotypes. Because the use of mAbs increases the specificity, accuracy and efficiency of diagnostic tests compared to polyclonal antisera, these two mAbs with different specificities are suitable for type-independent diagnosis of FMDV, such as DAS ELISA, or could be adapted to immuno-chromatographic or flow-through rapid test.     

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.     

Anti-idiotypic antibodies mimic bovine viral diarrhea virus antigen.

Polyclonal rabbit anti-idiotypic antibodies (anti-ids) against two neutralizing murine monoclonal antibodies (mAbs) specific to a bovine viral diarrhea virus (BVDV) glycoprotein, 53 kDa, were produced, purified, and characterized. Each anti-id inhibited the binding of its respective mAb to BVDV antigen in a competitive ELISA and blocked the immunoprecipitation of the 53 kDa protein by the mAb. The anti-ids also inhibited the virus-neutralizing activity of their homologous mAbs. These results suggest that the anti-ids bear an internal image of a BVDV antigen and mimic neutralizing epitopes on the 53 kDa protein. Treatment of MDBK cells with the anti-ids inhibited BVDV infection, indicating that they block a cellular component, such as a virus receptor, required for virus adsorption or entry. Inhibition of the homologous mAb and lack of inhibition of the heterologous mAb indicate that the anti-ids are specific for the unique antigen-binding sites on the mAbs.     

Generation and characterization of monoclonal antibodies to equine CD16

The low-affinity Fc receptor CD16 plays a central role in the inflammatory and innate immune responses of many species, but has not yet been investigated in the horse. Using the predicted extracellular region of equine CD16 expressed as a recombinant fusion protein with equine IL-4 (rIL-4/CD16), we generated a panel of mouse monoclonal antibodies (mAbs) that recognize equine CD16. Nine mAbs were chosen for characterization based upon recognition of CD16, but not IL-4, in ELISA. All nine mAbs recognized full-length, cell-surface CD16 expressed as a GFP fusion protein by CHO cells, but not the closely related Fc receptor CD32 expressed in the same system. In flow cytometric analysis with equine peripheral leukocytes, the mAbs labeled cells in the granulocyte, monocyte, and lymphocyte populations in a pattern consistent with other species. Monocytes that were strongly labeled with CD16 mAb 9G5 were also positive for the LPS receptor CD14. Cytospins made with peripheral leukocytes were immunohistochemically labeled and showed mAb recognition of primarily mononuclear cells. ELISA revealed that the nine mAbs can be grouped into three patterns of epitope recognition. These new antibodies will serve as useful tools in the investigation of equine immune responses and inflammatory processes.     

Characterisation of monoclonal antibodies to ovine interleukin-6 and the development of a sensitive capture ELISA

A purified recombinant ovine (rOv) interleukin-6 (IL-6) was used to generate specific murine monoclonal antibodies (mAbs) and a polyclonal rabbit antisera to this cytokine. From the 31 initial hybridoma cell lines generated, three stable clones were established which secreted mAbs to rOvIL-6, as judged by a direct enzyme-linked immunosorbent assay (ELISA) and Western blotting. Their specificity was further confirmed by demonstrating that none of the mAbs recognised any of the six other irrelevant recombinant ovine cytokines tested by direct ELISA. All three mAbs displayed cross-reactivity with human and African green monkey IL-6 as demonstrated by direct ELISA and Western blotting. In contrast, the polyclonal antibodies only cross-reacted with bovine IL-6 and not with either of the human or monkey homologues. By combining a mAb with the polyclonal antisera a sensitive, IL-6-specific, capture ELISA was developed that had a sensitivity of 150 pg/ml. This detection system was unequivocally validated by demonstrating that native OvIL-6 could be detected in efferent lymph draining from a stimulated popliteal lymph node. In addition, one of the mAbs was shown to allow the detection of OvIL-6 by intracellular cytokine staining and flow cytometry.     

Production of antibodies against chicken interferon-gamma: demonstration of neutralizing activity and development of a quantitative ELISA

Four monoclonal antibodies (mAbs) specific for chicken interferon-gamma (ChIFN-gamma) were generated by gene gun immunization and were utilized to develop a mAb-based capture ELISA specific for ChIFN-gamma. Each mAb reacted specifically with both baculovirus and Escherichia coli-derived recombinant ChIFN-gamma in ELISA and Western Blot analysis or natural ChIFN-gamma in immunofluorescence experiments. As determined by competition ELISAs, mAbs 3D5, 4C6 and 3A3 recognized the same or adjacent epitopes on the ChIFN-gamma molecule, whereas mAb 1E12 recognized a distant epitope. Moreover, this latter mAb was able to highly neutralize the biological activities of both recombinant and natural ChIFN-gamma as measured by inhibition of viral replication and macrophage activation. To improve the detection of ChIFN-gamma, a capture ELISA was developed using mAb 1E12 as capture antibody and biotinylated mAb 4C6 as detection antibody. In addition to being more rapid and easier to perform than classical cell-mediated immunity tests, this ELISA has excellent sensitivity and improved specificity. The use of a specific rabbit polyclonal serum as revealing antibody further increased the sensitivity of the detection down to 0.5ng/ml of ChIFN-gamma. This ELISA would provide a sensitive tool to measure the in vitro release of ChIFN-gamma by T-cells in response to specific recall antigen.     

应用ＥＬＩＳＰＯＴ试验测定重组卡介苗诱导的Ｔ细胞应答

应用免疫磁性分离技术去除免疫小鼠脾脏细胞中ＣＤ４^ 或ＣＤ8^ Ｔ细胞后，在酶免疫斑点试验中证实表达大肠杆菌ＭalE蛋白的重组卡介苗（rBCG.MalE)诱导的Ｔ细胞应答是ＣＤ４^ t细胞依赖的对MalE、PPD持异Ｔ细胞应答的动态分析结果表明，，rBCG.MalE、ＢＣＧ诱导的特异ＣＤ４^ T细胞应答存在Ｔh1/Th2平衡转换现象，即起始阶段为Ｔh1应答，一段时间后出现Ｔh2应答，并逐步形成Ｔh1/Th２混合应答。这些结果，分为枝杆菌Ｔ细胞应答规律提供了新的认识，同时，亦表明ＢＣＧ是优良的外源抗原表达与运送载体。     

Monoclonal antibodies against chicken interleukin-6

Monoclonal antibodies (mAb) were produced against a recombinant (r) chicken interleukin-6 (IL-6). Eight mAbs produced were tested for isotype; ability to inhibit recombinant forms of chicken (ch), human (h) and murine (m) IL-6; and recognition of rchIL-6 by Western immunoblotting. The mAb isotypes were represented by IgG1 (one), IgG2a (six) and IgG2b (one). In a mouse B9 hybridoma cell bioassay with rmIL-6, four mAbs effectively inhibited activity of rmIL-6. Further bioassays with the four mAbs at varying concentrations showed that two of these mAbs (1.20.7 and 1.26.4) were quite effective at inhibiting rmIL-6. Recombinant forms of ch, h and mIL-6 were all tested in a bioassay with the most potent inhibiting mAb (1.26.4), and this mAb was effective in inhibiting all three recombinant IL-6 proteins. Western immunoblotting revealed identification of the original IL-6 immunogen used for mAb production. Based upon inhibition of IL-6 activity in a standard bioassay and IL-6 recognition by Western immunoblotting, mAb 1.26.4 was judged the most useful antibody for future studies and applications.     

Epitope specificity of monoclonal antibodies to the N-protein of porcine reproductive and respiratory syndrome virus determined by ELISA with synthetic peptides

In an attempt to develop an alternate to ELISAs using recombinant N-proteins as antigen for the sero-diagnosis of porcine reproductive and respiratory syndrome virus (PRRSV) infections of pigs I have measured the binding of nine anti-N-protein mAbs, which had been previously generated by various investigators, to overlapping peptides encompassing amino acids 19-70 of the N-proteins of the North American prototype (VR2332) and the European prototype (Lelystad virus, LV) of PRRSV. I also measured the binding of the mAbs to HerdChek ELISA plates coated with recombinant N-protein. All mAbs bound in an indirect ELISA to some of the peptides whether the mAbs had previously been reported to recognize continuous or discontinuous epitopes, but with different specificity and titer. Three mAbs bound with high titer to different linear epitopes located in amino acid segments 23-33, 31-50 and 43-56 and also with similar high titers to HerdChek plates. mAb SDOW17 bound with high titer to HerdChek plates but poorly to any of the peptides. In contrast, four mAbs bound with broad specificity to peptides containing an epitope(s) in amino acid segment 30-48, but poorly, or not at all, to HerdChek ELISA plates. Thus, this epitope is missing on the antigens of the HerdChek ELISA or is destroyed during immobilization of the antigens on the plate. A mAb to the N-protein of the closely related mouse arterivirus lactate dehydrogenase-elevating virus bound to the same epitope. Abs that bound with broad specificity to an epitope(s) in the 30-50 amino acid segment were also detected by the peptide ELISA in sera of 25 field sera that were sero-positive in the HerdChek ELISA, but also in sera of pigs from two out of three herds tested that were sero-negative by this test.     

传染性法氏囊病病毒VP2单克隆抗体夹心ELISA检测试剂盒的研制

为研制传染性法氏囊病病毒（IBDV）快速检测试剂盒,用重组IBDV-VP2蛋白免疫BALB/c小鼠,制备免疫脾细胞,与SP2/0骨髓瘤细胞触合,获得3株稳定分泌抗VP2蛋白单克隆抗体（mAb）的杂交瘤细胞株,分别命名为1D11、2G8和2E5,抗体亚类分别为IgG1κ、IgG2bκ和IgG1κ。间接免疫荧光试验（IFA）证明,3株单抗均与VP2发生特异性反应。相加ELISA证明3株mAb识别VP2不同的抗原表位。在病毒中和试验中,1D11和2G8腹水对IBDV的中和效价分别为104和103,而2E5无中和活性。用亲和层析方法纯化1D11和2E5,分别作为包被抗体和标记抗体,建立了IBDV夹心ELISA检测方法,优化了试验条件,测定了其主要性能指标,对IBDV的最低检出量为102 TCID50/mL。用夹心ELISA、AGP和RT-PCR 3种方法同步检测8种试验样品,夹心ELISA与RT-PCR的检测结果一致,显著高于AGP方法。组装成的试剂盒,置于37℃保存7d、4℃保存6个月和-20℃保存24个月,其检测结果没有显著差异（P〉0.05）。     

Monoclonal antibodies specific for Campylobacter fetus lipopolysaccharides

Four monoclonal antibodies (mAbs) (M1357, M1360, M1823 and M1825) which reacted with Campylobacter fetus lipopolysaccharide (LPS) core region epitopes were produced and characterized. Reactivity of these mAbs with C. fetus core LPS epitopes was determined by enzyme-linked immunosorbent assay (ELISA) with whole cell proteinase K digests and phenol-water extracted LPS, and by immunoblotting with proteinase K digests. The specificities of the four mAbs were evaluated using an indirect ELISA. One of the mAbs reacted with 42 and three of the mAbs reacted with 41 of the 42 C. fetus strains examined. No reaction was observed between the four mAbs and 32 non-C. fetus bacteria tested, with the exception of one mAb with one organism. The four mAbs reacted with serotype A and B strains indicating the presence of shared epitopes in C. fetus LPS core oligosaccharides. The specificities of three mAbs previously produced to C. fetus LPS O-antigens (M1177, M1183 and M1194) were also evaluated and no reaction was observed with these mAbs and the 32 non-C. fetus bacteria tested. Strong immunofluorescence reactions were observed with the anti-O chain mAbs and selected C. fetus strains of the homologous serotype. These anti-LPS core oligosaccharide and anti-LPS O chain mAbs are highly specific for C. fetus and are potentially useful as immunodiagnostic reagents for detection, identification and characterization of C. fetus.     

A monoclonal antibody to equine interleukin-4

Interleukin-4 (IL-4) is secreted by T helper type 2 cells, mast cells, basophils and eosinophils. Detection of IL-4 can contribute the evaluation of cellular immune responses during infectious diseases, immunological disorders or vaccination. We used recombinant equine IL-4 to generate a monoclonal antibody (mAb) to equine IL-4. The mAb detected recombinant IL-4 in mammalian cells transfected with different plasmids containing IL-4 cDNA. After mitogen stimulation of equine peripheral blood mononuclear cells, an intracellular protein was recognized by the new mAb in 1–2% of lymphocytes using flow cytometric analysis. In the presence of the secretion blocker Brefeldin A, the protein accumulated and was detected in 4–8% of lymphocytes stimulated with phorbol 12-myristate 13-acetate and ionomycin. Double staining with the new mAb and T-cell or B-cell markers identified a subpopulation of CD4⁺ T-cells expressing the protein recognized by the mAb. In addition, the protein was detectable in cell culture supernatants of mitogen stimulated cells by ELISA when using the new mAb for coating of the plates and a polyclonal antiserum to equine IL-4 for detection. In conclusion, the new mAb detects equine IL-4 and can be used for intracellular staining and ELISA to measure this important cytokine.     

猪圆环病毒2型衣壳蛋白优势B细胞表位重组蛋白免疫原性研究

猪圆环病毒2型(PCV-2)衣壳蛋白(Cap)是研制基因工程疫苗和血清抗体检测方法的主要候选抗原。为筛选免疫原性强的Cap抗原表位,将定位在Cap上的6个B细胞线性表位的编码基因序列重新组合,命名为R1234、R1235、R1236、R2345、R2346和R3456,然后克隆至pET32a原核表达载体,转化到大肠埃希菌中进行诱导表达。通过对表达和纯化条件的优化,最终获得6个多抗原表位串联体的重组蛋白。Western blot和间接ELISA结果显示,6组重组表位蛋白均能被PCV-2阳性血清特异性识别,具有良好的反应原性。将其与完整Cap分别免疫Balb/c小鼠,检测免疫后血清中特异性抗体和细胞因子水平以及脾脏淋巴细胞中CD4^+和CD8^+T淋巴细胞的比例。结果表明,R2346组合刺激机体产生的特异性抗体和细胞因子IFN-γ、IL-4的水平明显高于其他组,脾脏淋巴细胞中CD4^+和CD8^+T淋巴细胞含量与对照组相比显著增加(P<0.05),说明该重组蛋白能够刺激体液免疫和细胞免疫反应。因此,筛选获得的R2346重组表位蛋白可以作为研制PCV-2多表位基因工程疫苗和血清抗体诊断方法的候选抗原,用于PCV-2的预防和控制工作中。     

Characterization of specific antibodies and the establishment of sandwich ELISA and ELISPOT systems for swine IL-4

We produced four monoclonal antibodies (mAb) and two polyclonal antibodies using the purified cytokine expressed in bacteria and characterized them. Specific binding of each of the mAb and polyclonal antibodies to recombinant swine IL-4 (rSwIL-4) purified from Escherichia coli and baculovirus was demonstrated in an indirect ELISA and/or in western blotting. We established a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring concentration of SwIL-4 in biological samples and established an enzyme-linked immunospot (ELISPOT) assay for detecting IL-4-secreting cells using a mAb and a polyclonal IgG from goat. The detection limit of the sandwich ELISA for SwIL-4 was 78 pg/ml. Using sandwich ELISA, SwIL-4 was detected in the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Mycoplasma hyopneumoniae and could quantitate in supernatants of mitogen-stimulated PBMC culture. The ELISPOT system is useful for the detection of IL-4 producing cells in swine PBMC culture.     

Generation and characterization of monoclonal antibodies to equine NKp46

The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize full-length NKp46 expressed on the surface of transfected CHO cells as a GFP fusion protein. The mAbs recognized a population of lymphocytes by flow cytometric analysis that was morphologically similar to NKp46+ cells in humans and cattle. In a study using nine horses, representative mAb 4F2 labeled 0.8-2.1% PBL with a mean fluorescence intensity consistent with gene expression data. MAb 4F2+ PBL were enriched by magnetic cell sorting and were found to express higher levels of NKP46 mRNA than 4F2- cells by quantitative RT-PCR. CD3-depleted PBL from five horses contained a higher percentage of 4F2+ cells than unsorted PBL. Using ELISA, we determined that the nine mAbs recognize three different epitopes. These mAbs will be useful tools in better understanding the largely uncharacterized equine NK cell population.     

Characterization of monoclonal antibodies to equine interleukin-10 and detection of T regulatory 1 cells in horses

Interleukin-10 (IL-10) terminates inflammatory immune responses and inhibits activation and effector functions of T-cells, monocytes, macrophages and dendritic cells. IL-10 has also been found to be a key cytokine expressed by subpopulations of regulatory T-cells. In this report, we describe the generation and characterization of three monoclonal antibodies (mAbs) to equine IL-10. The antibodies were found to be specific for equine IL-10 using different recombinant equine cytokine/IgG fusion proteins. Two of the anti-equine IL-10 mAbs were selected for ELISA to detect secreted IL-10 in supernatants of mitogen stimulated equine peripheral blood mononuclear cells (PBMC). The sensitivity of the ELISA for detecting secreted IL-10 was found to be around 200pg/ml. The production of intracellular IL-10 was measured in equine PBMC by flow cytometry. PBMC were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of the secretion blocker Brefeldin A. All three anti-IL-10 mAbs detected a positive population in PMA stimulated lymphocytes which was absent in the medium controls. Around 80% of the IL-10(+) cells were CD4(+). Another 15% were CD8(+) cells. Double staining with IL-4 or interferon-gamma (IFN-gamma) indicated that PMA and ionomycin stimulation induced 80% IL-10(+)/IFN-gamma(+) lymphocytes, while only 5% IL-10(+)/IL-4(+) cells were observed. By calculation, at least 60% of the IL-10(+)/IFN-gamma(+) cells were CD4(+) lymphocytes. This expression profile corresponds to the recently described T regulatory 1 (T(R)1) cell phenotype. In summary, the new mAbs to equine IL-10 detected native equine IL-10 by ELISA and flow cytometry and can be used for further characterization of this important regulatory cytokine in horses.