Evaluation of the reactivity of commercially available monoclonal antibodies with equine cytokines.
Abstract: Research on equine cytokines is often performed by analyses of mRNA. For many equine cytokines an analysis on the actual protein level is limited by the availability of antibodies against the targeted cytokines. Generation of new antibodies is ongoing but time consuming. Thus, testing the reactivity of commercially available antibodies for cross-reactivity with equine cytokines is of particular interest. Fifteen monoclonal antibodies against IL-1β, IL-6, IL-8, IL-12, IL-18 and Granulocyte Macrophage Colony stimulating factor (GM-CSF) of different species were evaluated for reactivity with their corresponding equine cytokines. Dot Blot (DB) and Western Blot (WB) analyses were performed using recombinant equine cytokines as positive controls. Immunohistochemistry (IHC) was carried out on equine tissue and flow cytometry on equine PBMC as positive controls. As expected, three equine IL-1β antibodies detected equine IL-1β in DB, WB and IHC. For these, reactivity in IHC has not been described before. One of them was also found to be suitable for intracellular staining of equine PBMC and flow cytometric analysis. Two antibodies raised against ovine GM-CSF cross-reacted with equine GM-CSF in DB, WB and IHC. For these anti-GM-CSF mAbs this is the first experimental description of cross-reactivity with equine GM-CSF (one mAb was predicted to be cross-reactive in WB in the respective data sheet). The other clone additionally proved to be appropriate in flow cytometric analysis. Two mAbs targeting porcine IL-18 cross-reacted in IHC, but did not show specificity in the other applications. No reactivity was shown for the remaining five antibodies in DB, although cross-reactivity of two of the antibodies was described previously. The results obtained in this study can provide beneficial information for choosing of antibodies for immunological tests on equine cytokines.
Copyright © 2013 Elsevier B.V. All rights reserved.
Publication Date: 2013-09-25 PubMed ID: 24139393DOI: 10.1016/j.vetimm.2013.09.012Google Scholar: Lookup
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- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article discusses the testing of commercially available monoclonal antibodies for reactivity with horse cytokines.
Objective and Significance of the Study
- The main objective of this study is to test the reactivity of commercially available antibodies, designed for different species, with equine (horse) cytokines – proteins essential for cell signaling.
- The significance of this research lies in its importance for understanding the immune system of horses, which frequently have been analyzed mostly on the genetic (mRNA) level due to lack of specific antibodies against many equine cytokines. With these findings, immunological research may be extended to the actual protein level.
Materials and Methods
- The researchers tested 15 different monoclonal antibodies directed against six different cytokines (IL-1β, IL-6, IL-8, IL-12, IL-18, and GM-CSF).
- Three different techniques were used in testing: Dot Blot (DB), Western Blot (WB), and Immunohistochemistry (IHC). Additionally, flow cytometry analysis was conducted on equine PBMC (Peripheral Blood Mononuclear Cells).
Main Findings and Outcomes
- The antibodies against three equine IL-1β cytokines showed successful detection in DB, WB, and IHC tests. One of them was also found suitable for intracellular staining of equine PBMC and flow cytometric analysis, representing a novelty for this research area.
- Two antibodies raised for ovine (sheep) GM-CSF cross-reacted with equine GM-CSF in all the three tests and showed their first experimental proof as cross-reactive with equine GM-CSF. Moreover, one of these antibodies also proved useful in flow cytometric analysis.
- Two antibodies targeting porcine (pig) IL-18 cytokine showed reactivity in IHC but failed to do so in other applications, emphasizing the importance of application-specific evaluation.
- No reactivity was observed for the remaining five antibodies in DB, despite the previous data mentioning their potential cross-reactivity.
- The results can provide potential guidance in selection of antibodies for equine immunological research.
Cite This Article
APA
Schnabel CL, Wagner S, Wagner B, Durán MC, Babasyan S, Nolte I, Pfarrer C, Feige K, Murua Escobar H, Cavalleri JM.
(2013).
Evaluation of the reactivity of commercially available monoclonal antibodies with equine cytokines.
Vet Immunol Immunopathol, 156(1-2), 1-19.
https://doi.org/10.1016/j.vetimm.2013.09.012 Publication
Researcher Affiliations
- University of Veterinary Medicine Hannover, Foundation, Clinic for Horses, Bünteweg 9, 30559 Hannover, Germany. Electronic address: Christiane.Schnabel@tiho-hannover.de.
MeSH Terms
- Animals
- Antibodies, Monoclonal / immunology
- Blotting, Western
- Cross Reactions
- Cytokines / analysis
- Cytokines / immunology
- Flow Cytometry
- Horses
- Immunohistochemistry
- Recombinant Proteins / immunology
Citations
This article has been cited 5 times.- Delcambre GH, Liu J, Herrington JM, Vallario K, Long MT. Immunohistochemistry for the detection of neural and inflammatory cells in equine brain tissue. PeerJ 2016;4:e1601.
- Hall SA, Stucke D, Morrone B, Lebelt D, Zanella AJ. Simultaneous detection and quantification of six equine cytokines in plasma using a fluorescent microsphere immunoassay (FMIA). MethodsX 2015;2:241-8.
- Schnabel CL, Steinig P, Koy M, Schuberth HJ, Juhls C, Oswald D, Wittig B, Willenbrock S, Murua Escobar H, Pfarrer C, Wagner B, Jaehnig P, Moritz A, Feige K, Cavalleri JM. Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent. BMC Vet Res 2015 Jun 23;11:140.
- Lu LL, Sun J, Lai JJ, Jiang Y, Bai LH, Zhang LD. Neuron-glial antigen 2 overexpression in hepatocellular carcinoma predicts poor prognosis. World J Gastroenterol 2015 Jun 7;21(21):6649-59.
- Underberg BA, Van der Vekens E, Drews B, Kaessmeyer S. Cyclooxygenase-2 and von Willebrand factor-an immunohistochemical study of the equine foot with and without laminitis, post-mortem perfused with paraffin oil. Front Vet Sci 2025;12:1673415.
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