Screening of anti-human leukocyte monoclonal antibodies for reactivity with equine leukocytes

Three hundred and seventy-nine monoclonal antibodies (mAbs) against various human CD molecules supplied to the HLDA8 animal homologues section (including four isotype controls) were analysed for cross-reactivity with equine leukocytes. First, flow cytometric identification of positively reacting mAbs was performed in one laboratory. Thereafter, a second round of flow cytometric evaluation was performed, involving three laboratories participating in the study. The first test-round indicated 17 mAbs as potentially positive. After the second round of flow cytometric analysis, 14 mAbs remained (directed against CD2, CD11a, CD18, CD44, CD45, CD49d, CD91, CD163 and CD172) where cross-reactivity was anticipated based on similarities between the human and equine staining pattern. Additionally, there was 1 mAb with weak likely positive reactivity, 12 mAbs with positive staining, which likely do not reflect valuable data, 5 mAbs with clear alternate expression pattern from that expected from humans, 5 mAbs with a questionable staining pattern itself, i.e. that was variable between the three labs, 32 mAbs with weak-positive expression and alternate staining pattern, and 279 negative mAbs (including the four isotype controls) were detected. In 31 cases, more appropriate target cells, such as thymocytes or stem cells, were not available for the screening. The results underline the value of this "cross-reactivity" approach for equine immunology. However, as only a few mAbs against leukocyte surface antigens reacted positively (approximately 4% of the mAbs submitted), the analysis of further anti-human mAbs and directed efforts to develop species-specific anti-CD mAb are still required.     

Further analysis of anti-human leukocyte mAbs with reactivity to equine leukocytes by two-colour flow cytometry and immunohistochemistry

We have reported on the reactivity of anti-human CD molecules with equine leukocytes by single-colour flow cytometry (this issue). The objectives of this additional study were to test for the reliability of the results obtained, and to obtain further information on the positive populations of lymphocytes. Two-colour flow cytometry and immunohistochemistry were performed, using many of the positive mAbs and a few questionable ones from the first part of the study. All mAbs analysed by two-colour flow cytometry could be confirmed to their previous designation as "positive" or "questionable". Most of the mAbs tested were effective in immunohistochemistry, supporting previous results. Examples of positive results will be presented and limitations of the study will be discussed briefly.     

Characterization of monoclonal antibodies recognizing immunoglobulin kappa and lambda chains in pigs by flow cytometry.

The existence of two types of the immunoglobulin (Ig) light chain in pigs was documented>30 years ago and has been confirmed by the cloning of porcine light chain genes homologous to human and murine Ig kappa (Igkappa) and Ig lambda (Iglambda). However, immunochemical reagents defining these two light chain isotypes have not been characterized. Here, we show that rabbit antisera specific for human Igkappa and Iglambda and certain anti-porcine light chain monoclonal antibodies (mAb) are useful in distinguishing light chain isotypes by flow cytometry (FCM). Porcine B cell lines L23 and L35 stained positive only with anti-human Iglambda antiserum and were negative when tested using anti-human Igkappa antiserum. While mAbs K139.3E1, 1G6 and 27.7.1 also tested positive on these cell lines, mAb 27.2.1 did not. Therefore, FCM was used to examine the hypothesis that K139.3E1, 1G6 and 27.7.1 are Iglambda-specific whereas mAb 27.2.1 recognizes the Igkappa chain in pigs. Double staining of peripheral blood mononuclear cells (PBMC) with pairs of anti-light chain mAbs and using cocktails of anti-light chain mAbs and anti-human polyclonal antiserum, confirmed this hypothesis with the exception that mAb K139.3E1 appears to recognize only a subset of Iglambda(+) B cells in most pigs. In summary, we identified two pan-specific anti-pig Iglambda mAbs, one anti-lambda mAb that recognizes a lambda-light chain subset and one anti-pig Igkappa mAb.     

A study by flow cytometry of lymphocytes in sow colostrum

Mammary secretions contain leucocytes which may be of value to the neonate. The cells obtained from sow colostrum (1 to 2·5 × 10⁶ ml⁻¹) are mainly lymphocytes (10 to 25 per cent) and epithelial cells (more than 20 per cent). In milk, there are few lymphocytes (0·5 to 2 per cent) and mostly alveolar epithelial cells. The study of lymphocytes in the mammary secretions of sows has been made difficult by the high proportion of epithelial cells, which could not be separated from lymphocytes, and by a high background in membrane immunofluorescence labelling. This paper describes a method for the study of the cells in the mammary secretions of sows by flow cytometry. It showed that 70 to 90 per cent of colostral lymphocytes were T lymphocytes, with T8 lymphocytes predominating over T4, and that the ratio T4/T8 was significantly lower in colostrum (0·57) than in blood (0·80). There were no lymphocytes expressing interleukin-2-receptors in the colostrum of sows.     

Antigenic characterization of IBDV field isolates by their reactivity with a panel of monoclonal antibodies

Recently Infectious Bursal Disease Virus isolates have been described in USA displaying an antigenic drift. Many of the new isolates were very virulent for chickens. In several European countries severe outbreaks of Gumboro disease have also been reported from vaccinated and non-vaccinated flocks. Since vaccinated SPF birds were shown to be protected against challenge infection with the new isolates under laboratory conditions, a more detailed investigation of the European isolates is wanted. The similarity between the European and US field situation got us to use a panel of monoclonal antibodies (MCAs) previously applied to characterize US strains for testing European isolates. An antigen capture ELISA has been carried out directly on bursa homogenates of chickens form the field. One European (F52/70) and two US (Var. E and GLS-5) strains have been included as reference viruses. From the results presented here it can be concluded that the European isolates (Netherlands, France, UK, Germany, Jugoslavia and Spain) did not undergo the same antigenic drift as the US strains. A more extensive analysis of the isolates will be done to elucidate their role for disease outbreaks.     

Counting absolute number of lymphocytes in quail whole blood by flow cytometry

In a previous study, we reported a new method for counting quail blood cells. After quail blood cells were stained with fluorescent lipophilic dye (DiOC6(3)), absolute counts of erythrocytes, granulocytes, and monocytes were obtained by means of flow cytometry (FC). The FC method has the potential for application to avian blood cells count; however, the method was unable to distinguish between lymphocytes and thrombocytes. In the present study, we improved the FC method to obtain separate counts of lymphocytes using DiOC5(3). After quail blood cells were stained with DiOC5(3), the cells were measured with FC. Each blood cell type was distinguished by means of their typical FL-1 (green fluorescence) and SSC (side scatter). Absolute numbers of erythrocytes, granulocytes, monocytes and lymphocytes in whole blood were obtained. The improved FC analysis worked equally well with chicken (Gallus gallus) and goose (Anser cygnoides) blood.     

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.     

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.     

Detection of antibodies to equine arteritis virus by a monoclonal antibody-based blocking ELISA.

A potent ELISA antigen was prepared from equine arteritis virus (EAV) by differential centrifugation of EAV-infected cell culture fluid, followed by solubilization of the preparation by Triton X-100 treatment. Using this antigen and a mouse monoclonal antibody against the G(L) protein of EAV, a reliable blocking ELISA (bELISA) was developed for the detection of EAV antibodies in equine sera. The bELISA was evaluated using a total of 837 test serum samples. The relative sensitivity (n = 320) of the bELISA compared to the serum neutralization (SN) test was 99.4%. The bELISA appears to be a highly specific test, the specificity of which did not appear to be adversely affected by previous exposure of horses to non-EAV-containing biologicals. Of 119 serum samples, 21 from horses without any history of exposure to EAV and 98 from racetrack Thoroughbreds, 118 were negative in the SN test and bELISA. One sample was SN-negative but suspicious with the bELISA. Based on testing 465 SN-negative field samples and 52 SN-negative samples from experimental horses, and excluding any sera giving a suspicious reaction, the relative specificity of the bELISA was 97.7%. Samples should be examined undiluted and diluted 1/10 in the bELISA because the testing of sera of high neutralizing antibody titer may be affected by a prozone-like phenomenon. The bELISA is a more rapid and cost-efficient test than the SN test for the detection of EAV antibodies in equine sera.     

抗马动脉炎病毒N蛋白单克隆抗体的制备

为制备抗马动脉炎病毒(EAV)衣壳蛋白(N)的单克隆抗体(MAb),本研究通过原核表达重组N蛋白,纯化后免疫6周龄雌性BALB/c小鼠,细胞融合后经间接ELISA筛选获得两株能够稳定分泌抗EAV N蛋白的杂交瘤细胞株,MAbs亚型鉴定为IgG1,轻链为κ链。Western blot结果显示,这两株杂交瘤细胞分泌的MAb均能够识别EAV。EAV N蛋白MAb的制备,为EAV血清学检测方法的建立奠定了基础。     

Immunoprecipitation of equine CD molecules using anti-human MABs previously analyzed by flow cytometry and immunohistochemistry

Earlier studies investigating the cross-reactivity of antibodies submitted to the HLDA8 had used flow cytometry as a method of choice to screen mAbs for reactivity with equine leukocytes, including two-color flow-cytometry to characterize the lymphocyte population they detect. In addition, immuno-histochemistry (IHC) was used to detect distribution of positive cells in lymphoid tissue sections. In this study we performed immunoprecipitation (IP) to complement the previous results and add valuable information regarding the molecules detected by the cross-reacting antibodies. Surface molecules from primary equine PBMC or the equine cell line T8888 were biotinylated prior to precipitation to determine the molecular weight of the corresponding molecules in a western blot using streptavidin-AP. 21 out of 24 mAbs precipitated the molecules with a MW corresponding to its human orthologue. Positive mAbs were directed against CD2, CD5, CD11a, CD11b, CD14, CD18, CD21, CD44, CD83, CD91, CD172a, MHCI and MHCII. Three mAbs directed against CD49d, CD163, and CD206 which were unambiguously identified earlier by flow cytometry failed to immunoprecipitate the corresponding CD molecule. MAbs detecting CD molecules which are expressed internally like CD68 and mAbs of IgM class could not be included into this approach.     

Negative enrichment of bovine T lymphocytes with monoclonal antibodies and magnetic microspheres

A highly enriched population of bovine T lymphocytes was produced from peripheral blood leukocytes following the depletion of monoclonal antibody-labelled B lymphocytes and monocytes with magnetic microspheres. This negative-enrichment protocol was simple, rapid, and specific. Also, it had a high recovery efficiency and was consistently reproducible. The enriched T lymphocytes proliferated in response to recombinant bovine interleukin 2 and, following the addition of monocytes, to concanavalin A. This methodology made it possible to determine the proliferative responses of peripheral blood lymphocytes utilizing a constant number of T lymphocytes within each assay. In this way, the in vitro T lymphocyte responses were determined independent of changes in the number of responder cells within peripheral blood.     

Use of flow cytometry to develop and characterize a set of monoclonal antibodies specific for rabbit leukocyte differentiation molecules

Flow cytometry was used to identify and characterize monoclonal antibodies (mAbs) that react with rabbit leukocyte differentiation molecules (LDM). Screening sets of mAbs, developed against LDM in other species, for reactivity with rabbit LDM yielded 11 mAbs that recognize conserved epitopes on rabbit LDM orthologues and multiple mAbs that recognize epitopes expressed on the major histocompatibility class I or class II molecules. Screening of mAbs submitted to the Animal Homologues Section of the Eighth Human Leukocyte Differentiation Workshop yielded 7 additional mAbs. Screening of mAbs generated from mice immunized with leukocytes from rabbit thymus or spleen or concanavalin A activated peripheral blood and/or spleen lymphocytes has yielded 42 mAbs that recognize species restricted epitopes expressed on one or more lineages of leukocytes. Screening of the anti-rabbit mAbs against leukocytes from other species yielded one additional mAb. The studies show that screening of existing sets of mAbs for reactivity with rabbit LDM will not be productive and that a direct approach will be needed to develop mAbs for research in rabbits. The flow cytometric approach we developed to screen for mAbs of interest offers a way for individual laboratories to identify and characterize mAbs to LDM in rabbits and other species. A web-based program we developed provides a source of information that will facilitate analysis. It contains a searchable data base on known CD molecules and a data base on mAbs, known to react with LDM in one or more species of artiodactyla, equidae, carnivora, and or lagomorpha.     

Protective immunity to Ascaris suum: analysis of swine peripheral blood cell subsets using monoclonal antibodies and flow cytometry

Outbred domestic swine or SLA inbred miniature swine were exposed to Ascaris suum either naturally on contaminated lots or by inoculation with UV-irradiated attenuated eggs. Both inbred and outbred swine developed virtually complete protection to a challenge of 10 000 eggs after natural exposure, but inbred swine were less resistant than outbred swine after UV-egg exposure. Flow cytometric analysis of peripheral blood mononuclear cells from these animals, performed to determine changes in cell subsets including helper T-cells, cytotoxic/suppressor T-cells, macrophages, and cells expressing class II major histocompatibility antigens, showed that both outbred and inbred swine had similar responses after parasite exposure. The levels of helper T-cells and cytotoxic/suppressor T-cells did not change after parasite exposure, while there was an appreciable but transient increase in macrophages only in those swine naturally exposed to A. suum. Swine exposed to A. suum, both naturally and by inoculation with UV-eggs, showed an increase in the amount of class II antigens detectable per cell. In a second set of experiments, outbred swine were exposed to A. suum naturally or by repeated experimental inoculation with different doses of normal eggs, and protective immunity and changes in blood cell subsets were determined. The greatest change in blood cell subsets was found at 3 and 5 weeks after initial parasite exposure, when macrophages were elevated moderately in a group of pigs inoculated every other day with 1000 eggs and markedly in a group that was naturally exposed; class II antigen expression was also increased during this period. These increases preceded peak serum antibody responses, which were lower in the naturally-exposed group relative to the experimentally-inoculated group. Both groups had high levels of protective immunity. This suggests than natural exposure to A. suum may activate cells and enhance specific immune responses to give high levels of protection.     

Production and characterisation of two monoclonal antibodies recognising equine IgG Fc receptors

Despite the importance of IgG Fc receptors in the regulation of various immunological mechanisms, these receptors have not been well characterised in the domesticated animals including equines. This paper describes the production of two monoclonal antibodies (CVS 59 and CVS 61) that recognise equine IgG Fc receptors. Fusions were conducted using BALB/c mice hyperimmunised with equine peripheral blood mononuclear cells. Hybridoma supernatants were screened on the basis of their ability to inhibit the rosetting of equine antibody coated sheep erythrocytes with equine peripheral blood mononuclear cells. The mAbs were then characterised for cellular distribution of the antigen by flow cytometry. Using immunoprecipitation, it was shown that both under reducing and non-reducing conditions, CVS 59 and CVS 61 precipitated a 80 and 40 kD protein, respectively. From the functional study coupled with cellular distribution and molecular weight of the antigen recognised by these mAbs, it would appear that CVS 59 and CVS 61 recognise an epitope on equine equivalent of human and murine FcgammaRIII and FcgammaRII, respectively.     

Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink.     

Evaluation of the suitability of monoclonal antibodies for flow cytometric intracellular cytokine detection in porcine peripheral blood lymphocytes

Panels of monoclonal antibodies (mAbs) specific for porcine interleukin (IL)-2, IL-4, IL-6, IL-12, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha were evaluated for their applicability in flow cytometric intracellular cytokine detection. Peripheral blood mononuclear cells were short-time stimulated in the presence of brefeldin-A, ionomycin and phorbol-12-myristate-13-acetate, fixed and incubated with the respective mAbs as well as phycoerythrin-conjugated second-step antibodies. Suitability of mAbs was judged by use of statistical data and by visual control of scattergrams, considering the capability of mAbs to discriminate between cytokine-positive and cytokine-negative cell populations. The number of suitable clones differed to a high degree between the investigated cytokines, but at least one mAb fitting for flow cytometric intracellular cytokine detection could be identified within each panel. Monoclonal Abs producing scattergrams with a clear demarcation between positive and negative cell populations were found within the anti-IL-2, IL-6 and IFN-gamma panels, whereas less well defined positive and negative cell populations could be generated by use of mAbs within the anti-IL-4 and TNF-alpha panels. Only one moderately fitting mAb was identified within the anti-IL-12 panel. After having evaluated the best fitting mAbs, these were used to obtain reference levels for the physiological range of porcine lymphocytic cytokine production in a second set of experiments. For that reason, 13 clinically healthy pigs aged between 6 weeks and 6 months were investigated. Data presented are given as mean +/- SD of the percentage of positive-staining lymphocytes: IL-2, 45.5 +/- 27.6; IL-4, 34.1 +/- 21.3; IL-6, 45.4 +/- 23.8; IL-12, 13.9 +/- 8.6; TNF-alpha, 43.4 +/- 11.3; IFN-gamma, 65.5 +/- 14.8.     

马传染性贫血病毒基质蛋白p15单克隆抗体的制备

为制备抗马传染性贫病毒(EIAV)的单克隆抗体(MAb),本研究用纯化的重组EIAV基质蛋白作为免疫原免疫BALB/c小鼠,采用淋巴细胞杂交瘤技术,建立了抗EIAV基质蛋白p15抗体的杂交瘤细胞.应用重组p15和EIAV总蛋白为抗原建立的ELISA以及EIAV感染细胞的间接免疫荧光法,对杂交瘤细胞进行了有限稀释法筛选,获得了7株稳定分泌抗p15抗体的杂交瘤细胞株,分别命名为:1F12、1E2、1E3、4B10、4H7、5E5、4G5.这些抗p15 MAb均能与感染驴胎皮肤细胞的EIAV和经SDS-PAGE分离的EIAV总蛋白中的相应蛋白结合.抗体效价叠加实验表明,这7株MAb中含有至少3种识别不同抗原决定簇的抗体.这些p15 MAb的获得有助于对EIAV和慢病毒的深入研究.     

Sexing of sperm by flow cytometry

Economics dictate that livestock producers will be under increasing pressure to optimise output. A technique for sex pre-selection could help by reducing the number of females required to produce a given number of progeny of the desired sex; the technique would be particularly useful to the dairy industry. Live mammalian sperm, stained with a vital dye and analysed by flow cytometry, show a bimodal fluorescence distribution. Such bimodality may represent two overlapping subpopulations of X- and Y-chromosome bearing sperm. To test this hypothesis, sperm from the two subpopulations were separated using the sorting capacity of a flow cytometer and were used for the insemination of suitably prepared females. The sex of the resulting progeny was determined either by anatomical criteria or by identification of the sex chromosomes by karyotyping. Insufficient data are available so far to provide statistically significant evidence in support of the hypothesis, but a preliminary sequential analysis indicates a progressive tendency towards significance.