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

Department of Clinical Sciences, Colorado State University, Fort Collins, Colorado, United States
Veterinary Immunology and Immunopathology (Impact Factor: 1.54). 10/2007; 119(1-2):63-80. DOI: 10.1016/j.vetimm.2007.06.034
Source: PubMed


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.

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    • "Evaluation of cross-species reactivity of commercially available antibodies, particularly CD antigens, has been largely performed in equine tissues prepared for whole-cell analysis like flow-cytometry (Johne et al., 1997;Mérant et al., 2003;Terio et al., 2003;Kunisch et al., 2004;Ibrahim et al., 2007) or on fresh or frozen specimens (Bilzer et al., 1995;Zeng et al., 1996;Lemos et al., 2008;Härtig et al., 2009). Large screenings of non-equine derived antibodies have often resulted in limited identification of equine reactive reagents (Ibrahim et al., 2007;Schnabel et al., 2013;Szabo & Gulya, 2013). Of the 26 antibodies in this study, six antibodies successfully reacted with FFPE equine tissues. "
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    ABSTRACT: Phenotypic characterization of cellular responses in equine infectious encephalitides has had limited description of both peripheral and resident cell populations in central nervous system (CNS) tissues due to limited species-specific reagents that react with formalin-fixed, paraffin embedded tissue (FFPE). This study identified a set of antibodies for investigating the immunopathology of infectious CNS diseases in horses. Multiple commercially available staining reagents and antibodies derived from antigens of various species for manual immunohistochemistry (IHC) were screened. Several techniques and reagents for heat-induced antigen retrieval, non-specific protein blocking, endogenous peroxidase blocking, and visualization-detection systems were tested during IHC protocol development. Boiling of slides in a low pH, citrate-based buffer solution in a double-boiler system was most consistent for epitope retrieval. Pressure-cooking, microwaving, high pH buffers, and proteinase K solutions often resulted in tissue disruption or no reactivity. Optimal blocking reagents and concentrations of each working antibody were determined. Ultimately, a set of monoclonal (mAb) and polyclonal antibodies (pAb) were identified for CD3 + (pAb A0452, Dako) T-lymphocytes, CD79αcy + B-lymphocytes (mAb HM57, Dako), macrophages (mAb MAC387, Leica), NF-H + neurons (mAb NAP4, EnCor Biotechnology), microglia/macrophage (pAb Iba-1, Wako), and GFAP + astrocytes (mAb 5C10, EnCor Biotechnology). In paraffin embedded tissues, mAbs and pAbs derived from human and swine antigens were very successful at binding equine tissue targets. Individual, optimized protocols are provided for each positively reactive antibody for analyzing equine neuroinflammatory disease histopathology.
    Full-text · Article · Jan 2016 · PeerJ
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    • "Greater than 90% of the analyzed PBMC were CD172a+, indicating that they were monocytes/macrophages (data not shown). Although the anti-CD172a mAb also identifies equine granulocytes [40], the ficoll hypaque PBMC isolation protocol (which removes the majority of granulocytes) together with the lack of internal complexity in the labeled cells (data not shown), provided further evidence that they were monocytes/macrophages. Overall, these data confirmed the SCID phenotype and indicated that the establishment of T. equi infection in SCID1 and SCID2 did not involve B or T lymphocytes. "
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    ABSTRACT: Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.
    Full-text · Article · Oct 2013 · PLoS ONE
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    • "According to Borjesson and Peroni (2011), the capacity to characterize equine MSCs is hampered by the limited availability of commercial antibodies and by variability in cross-reactions . Ibrahim et al. (2007) showed that of 379 human CD antibodies, only 14 (4%) showed cross-reactivity with equine leukocytes. Alternatively, De Schauwer et al. (2011) suggested the use of a panel of cell surface markers for equine MSCs. "
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    ABSTRACT: The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium-rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n = 64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies. Microsc. Res. Tech., 2013. © 2013 Wiley Periodicals, Inc.
    Full-text · Article · Mar 2013 · Microscopy Research and Technique
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