Monoclonal antibodies to equine CD14.
Abstract: CD14 is a receptor for the complex of lipopolysaccaride (LPS) and LPS-binding protein. Binding of this complex to CD14 in association with Toll-like receptor 4 provides a major pathway for the initiation of innate immune responses to bacterial pathogens. We used a mammalian expressed extracellular region of equine CD14 (rCD14) derived from an IgG fusion protein to produce monoclonal antibodies (mAbs) to CD14. Eight mAbs were tested by flow cytometric analysis of equine leukocytes and by immunoblotting using rCD14 indicating that the mAbs recognized at least three different epitopes on equine CD14. One mAb, clone 105, was used for further characterization of CD14+ cells in peripheral blood mononuclear cells (PBMC). Phenotyping indicated that the majority of the CD14+ PBMC were non-B/non-T-cells. Magnetic cell sorting enriched CD14+ cells to > 95% as detected by flow cytometry. Differential cell counts on Wright's-stained cytospin smears of CD14+ cell fractions demonstrated that 49-73% of them were monocytes. The discrepancy between CD14+ cells detected by flow cytometric analysis and monocytes based on morphologic criteria suggests that some of the equine CD14+ PBMC are lymphoid cells. The mAbs to equine CD14 provide new tools for cellular analysis and CD14+ cell isolation in horses.
Copyright © 2010 Elsevier B.V. All rights reserved.
Publication Date: 2010-07-31 PubMed ID: 20674042DOI: 10.1016/j.vetimm.2010.07.003Google Scholar: Lookup
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- Journal Article
- Research Support
- U.S. Gov't
- Non-P.H.S.
Summary
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This research study revolves around the production of monoclonal antibodies to CD14 in horses. The CD14 receptor plays a significant role in initiating immune responses to bacterial pathogens. The antibodies produced were tested to understand their reaction with different epitopes on CD14. The study also involved the increased purification and identification of CD14+ cells within peripheral blood mononuclear cells (PBMC).
Production of Monoclonal Antibodies
- The study used a mammalian expressed extracellular region of equine CD14 (rCD14) derived from an IgG fusion protein to produce monoclonal antibodies (mAbs).
- These antibodies were then tested using flow cytometric analysis and immunoblotting on equine leukocytes.
- The results indicated that the antibodies recognized at least three different epitopes on equine CD14.
Characterization of CD14+ Cells
- One of the monoclonal antibodies, known as clone 105, was further used to identify the characteristics of CD14+ cells within peripheral blood mononuclear cells (PBMC).
- Phenotyping suggested that majority of these CD14+ cells in PBMC were neither B cells nor T cells.
Magnetic Cell Sorting and Cell Counting
- The researchers then used magnetic cell sorting to purify CD14+ cells, attaining a purity level of over 95%.
- Differential cell counts were carried out on Wright’s-stained cytospin smears of these CD14+ cells.
- The cell counts suggested that about 49-73% of these cells were monocytes.
Discrepancy in CD14+ Cell Detection
- There was a discrepancy in the detection of CD14+ cells based on flow cytometry analysis and morphological observation of monocytes.
- This led to a proposal that some equine CD14+ PBMC might be lymphoid cells.
Conclusion
- The produced monoclonal antibodies to the equine CD14 have presented new tools for cellular analysis in horses.
- Further, the antibodies can be employed in the isolation of CD14+ cells, advancing our understanding of these cells and their role in the equine immune response.
Cite This Article
APA
Kabithe E, Hillegas J, Stokol T, Moore J, Wagner B.
(2010).
Monoclonal antibodies to equine CD14.
Vet Immunol Immunopathol, 138(1-2), 149-153.
https://doi.org/10.1016/j.vetimm.2010.07.003 Publication
Researcher Affiliations
- Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
MeSH Terms
- Animals
- Antibodies, Monoclonal / biosynthesis
- Antibody Specificity
- Base Sequence
- DNA Primers / genetics
- Epitopes / genetics
- Epitopes / immunology
- Flow Cytometry
- Horses / blood
- Horses / genetics
- Horses / immunology
- Immunity, Innate
- Immunophenotyping
- Leukocytes, Mononuclear / immunology
- Lipopolysaccharide Receptors / blood
- Lipopolysaccharide Receptors / genetics
- Lipopolysaccharide Receptors / immunology
- Mice
- Mice, Inbred BALB C
- Recombinant Fusion Proteins / genetics
- Recombinant Fusion Proteins / immunology
Citations
This article has been cited 17 times.- Simonin EM, Wagner B. IgE-binding monocytes upregulate the coagulation cascade in allergic horses.. Genes Immun 2023 Jun;24(3):130-138.
- Keller LE, Tait Wojno ED, Begum L, Fortier LA. T Helper 17-Like Regulatory T Cells in Equine Synovial Fluid Are Associated With Disease Severity of Naturally Occurring Posttraumatic Osteoarthritis.. Am J Sports Med 2023 Mar;51(4):1047-1058.
- Gressler AE, Lübke S, Wagner B, Arnold C, Lohmann KL, Schnabel CL. Comprehensive Flow Cytometric Characterization of Bronchoalveolar Lavage Cells Indicates Comparable Phenotypes Between Asthmatic and Healthy Horses But Functional Lymphocyte Differences.. Front Immunol 2022;13:896255.
- Kang H, Bienzle D, Lee GKC, Piché É, Viel L, Odemuyiwa SO, Beeler-Marfisi J. Flow cytometric analysis of equine bronchoalveolar lavage fluid cells in horses with and without severe equine asthma.. Vet Pathol 2022 Jan;59(1):91-99.
- Raza F, Babasyan S, Larson EM, Freer HS, Schnabel CL, Wagner B. Peripheral blood basophils are the main source for early interleukin-4 secretion upon in vitro stimulation with Culicoides allergen in allergic horses.. PLoS One 2021;16(5):e0252243.
- Larson EM, Babasyan S, Wagner B. IgE-Binding Monocytes Have an Enhanced Ability to Produce IL-8 (CXCL8) in Animals with Naturally Occurring Allergy.. J Immunol 2021 May 15;206(10):2312-2321.
- Hagen A, Lehmann H, Aurich S, Bauer N, Melzer M, Moellerberndt J, Patané V, Schnabel CL, Burk J. Scalable Production of Equine Platelet Lysate for Multipotent Mesenchymal Stromal Cell Culture.. Front Bioeng Biotechnol 2020;8:613621.
- Patel RS, Tomlinson JE, Divers TJ, Van de Walle GR, Rosenberg BR. Single-cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse cell types including T-bet(+) B cells.. BMC Biol 2021 Jan 22;19(1):13.
- Menarim BC, Gillis KH, Oliver A, Ngo Y, Werre SR, Barrett SH, Rodgerson DH, Dahlgren LA. Macrophage Activation in the Synovium of Healthy and Osteoarthritic Equine Joints.. Front Vet Sci 2020;7:568756.
- Larson EM, Babasyan S, Wagner B. Phenotype and function of IgE-binding monocytes in equine Culicoides hypersensitivity.. PLoS One 2020;15(5):e0233537.
- Schwab UE, Tallmadge RL, Matychak MB, Felippe MJB. Effects of autologous stromal cells and cytokines on differentiation of equine bone marrow-derived progenitor cells.. Am J Vet Res 2017 Oct;78(10):1215-1228.
- Ziegler A, Everett H, Hamza E, Garbani M, Gerber V, Marti E, Steinbach F. Equine dendritic cells generated with horse serum have enhanced functionality in comparison to dendritic cells generated with fetal bovine serum.. BMC Vet Res 2016 Nov 15;12(1):254.
- Sarkar S, Chelvarajan L, Go YY, Cook F, Artiushin S, Mondal S, Anderson K, Eberth J, Timoney PJ, Kalbfleisch TS, Bailey E, Balasuriya UB. Equine Arteritis Virus Uses Equine CXCL16 as an Entry Receptor.. J Virol 2016 Jan 13;90(7):3366-84.
- Yeo WM, Osterrieder N, Stokol T. Equine herpesvirus type 1 infection induces procoagulant activity in equine monocytes.. Vet Res 2013 Mar 11;44(1):16.
- Cavatorta DJ, Erb HN, Felippe MJ. Activation-induced FoxP3 expression regulates cytokine production in conventional T cells stimulated with autologous dendritic cells.. Clin Vaccine Immunol 2012 Oct;19(10):1583-92.
- Noronha LE, Harman RM, Wagner B, Antczak DF. Generation and characterization of monoclonal antibodies to equine CD16.. Vet Immunol Immunopathol 2012 Apr 15;146(2):135-42.
- Hackett CH, Flaminio MJ, Fortier LA. Analysis of CD14 expression levels in putative mesenchymal progenitor cells isolated from equine bone marrow.. Stem Cells Dev 2011 Apr;20(4):721-35.
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