Immunoprecipitation of equine CD molecules using anti-human MABs previously analyzed by flow cytometry and immunohistochemistry.
Abstract: 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.
Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.
Publication Date: 2011-08-04 PubMed ID: 22070824DOI: 10.1016/j.vetimm.2011.07.021Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research investigated the effectiveness of using anti-human monoclonal antibodies (mAbs) to detect equine (horse) cell surface molecules, using a technique known as immunoprecipitation. This followed previous research that used flow cytometry and immunohistochemistry. The study results found that most mAbs successfully precipitated the corresponding equine molecules, providing additional information for previous research.
Research Overview
- The study performed immunoprecipitation, a method used for investigating protein-protein interactions, to complement and expand upon previous research results.
- The previous studies used flow cytometry and immuno-histochemistry to screen for reactivity of mAbs with equine leukocytes.
- In this study, surface molecules from primary equine peripheral blood mononuclear cells (PBMC) or the equine cell line T8888 were treated with biotin before precipitation, to understand the molecular weight of the detected molecules.
Study Findings
- Using immunoprecipitation, the study found successful precipitation and detection of 21 out of 24 mAbs. The precipitated molecules had a molecular weight corresponding to their human counterparts.
- The successfully detected CD molecules were CD2, CD5, CD11a, CD11b, CD14, CD18, CD21, CD44, CD83, CD91, CD172a, MHCI and MHCII.
- Three mAbs (CD49d, CD163, and CD206) previously identified by flow cytometry failed to immunoprecipitate their corresponding CD molecules.
Limited Scope
- The researchers noted that their approach could not be applied to all mAbs. Specifically, it could not be used for mAbs recognizing CD molecules that are expressed internally, like CD68, or for mAbs of the class IgM.
Research Significance
- This research provides additional information toward understanding cross-reactivity of mAbs with equine leukocytes. The results can contribute to advancing immunological research in equines, with potential applications in veterinary health and disease research.
Cite This Article
APA
Ibrahim S, Steinbach F.
(2011).
Immunoprecipitation of equine CD molecules using anti-human MABs previously analyzed by flow cytometry and immunohistochemistry.
Vet Immunol Immunopathol, 145(1-2), 7-13.
https://doi.org/10.1016/j.vetimm.2011.07.021 Publication
Researcher Affiliations
- Institute for Zoo and Wildlife Research (IZW), Alfred Kowalke Str. 17, 10315 Berlin, Germany.
MeSH Terms
- Animals
- Antibodies, Monoclonal / immunology
- Antigens, CD / immunology
- Biomarkers
- CD2 Antigens / immunology
- CD5 Antigens / immunology
- Cell Line
- Cross Reactions / immunology
- Flow Cytometry / veterinary
- Horses
- Humans
- Immunohistochemistry / veterinary
- Immunoprecipitation / methods
- Immunoprecipitation / veterinary
- Leukocytes / cytology
- Leukocytes / immunology
Citations
This article has been cited 8 times.- 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.
- Merlo B, Teti G, Mazzotti E, Ingrà L, Salvatore V, Buzzi M, Cerqueni G, Dicarlo M, Lanci A, Castagnetti C, Iacono E. Wharton's Jelly Derived Mesenchymal Stem Cells: Comparing Human and Horse.. Stem Cell Rev Rep 2018 Aug;14(4):574-584.
- Tallmadge RL, Miller SC, Parry SA, Felippe MJB. Antigen-specific immunoglobulin variable region sequencing measures humoral immune response to vaccination in the equine neonate.. PLoS One 2017;12(5):e0177831.
- Prieto JMB, Tallmadge RL, Felippe MJB. Developmental expression of B cell molecules in equine lymphoid tissues.. Vet Immunol Immunopathol 2017 Jan;183:60-71.
- Badial PR, Tallmadge RL, Miller S, Stokol T, Richards K, Borges AS, Felippe MJ. Applied Protein and Molecular Techniques for Characterization of B Cell Neoplasms in Horses.. Clin Vaccine Immunol 2015 Nov;22(11):1133-45.
- Kol A, Wood JA, Carrade Holt DD, Gillette JA, Bohannon-Worsley LK, Puchalski SM, Walker NJ, Clark KC, Watson JL, Borjesson DL. Multiple intravenous injections of allogeneic equine mesenchymal stem cells do not induce a systemic inflammatory response but do alter lymphocyte subsets in healthy horses.. Stem Cell Res Ther 2015 Apr 15;6(1):73.
- Gittel C, Brehm W, Burk J, Juelke H, Staszyk C, Ribitsch I. Isolation of equine multipotent mesenchymal stromal cells by enzymatic tissue digestion or explant technique: comparison of cellular properties.. BMC Vet Res 2013 Oct 29;9:221.
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