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Home / Equine Research Bank / Vet Immunol Immunopathol / Development of a bead-based multiplex assay for simultaneous...
Veterinary immunology and immunopathology2008; 127(3-4); 242-248; doi: 10.1016/j.vetimm.2008.10.313

Development of a bead-based multiplex assay for simultaneous quantification of cytokines in horses.

Abstract: The detection and quantification of equine cytokines has been hampered by the lack of antibodies for many years. With the development of antibody pairs for equine cytokines during the past years, the quantification of these essential regulators of the immune response became possible. After being successfully tested by enzyme-linked immunosorbent assays (ELISA), three of these anti-cytokine reagents were used here to establish the first cytokine multiplex assay for equine IL-4, IL-10 and IFN-alpha. A fluorescent bead-based system was used as matrix for this assay that allows the simultaneous detection of the cytokines in a single sample by a Luminex analyzer. Equine recombinant cytokine/IgG fusion proteins were validated as standards for quantification of the individual cytokines. The analytical sensitivities of the multiplex assay were found to be 40 pg/ml for IL-4 and 15 pg/ml for IL-10 and IFN-alpha. The sensitivity of cytokine detection by the multiplex assay was increased by 13- to 150-fold compared to the corresponding ELISA. The specificity of the multiplex assay was validated using cell culture supernatants from equine peripheral blood mononuclear cells (PBMC) stimulated with different mitogens or infected with equine herpesvirus type 1 (EHV-1). As predicted, supernatants from PBMC stimulated with different mitogens contained IL-4 and IL-10, but no IFN-alpha. EHV-1 infection of PBMC resulted in a dose-dependent secretion of IFN-alpha. Low concentrations of IL-10 were also measured. IL-4 was not detectable in these samples. The resulting detection pattern found for the multiplex analysis and assays performed with individual standard cytokines indicated that individual bead assays did not interfere or cross-react during simultaneous detection of equine IL-4, IL-10 and IFN-alpha. The equine cytokine multiplex assay is a valuable and cost-effective tool for quantification of IL-4, IL-10 and IFN-alpha and can be used for manifold immunological applications. In the future, the assay can also be expanded by adding bead assays for other equine cytokines and chemokines to the existing platform.
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Publication Date: 2008-10-18 PubMed ID: 19027964DOI: 10.1016/j.vetimm.2008.10.313Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research presents the creation of a bead-based multiplex assay that can quantify three cytokines (IL-4, IL-10, and IFN-alpha) simultaneously in horses. The method increases the sensitivity of cytokine detection, developed due to advancements in anti-cytokine reagents and is particularly beneficial in terms of being cost-effective.

Development of the Multiplex Assay

  • The researchers utilized the development of antibody pairs for equine cytokines that have happened in the recent past to quantify these vital regulators of immune response. Prior to this, detection and quantification were challenging due to a lack of necessary antibodies.
  • Three anti-cytokine reagents which were previously and successfully tested by enzyme-linked immunosorbent assays (ELISAs) were used in the development of this cytokine multiplex assay, targeted to equine IL-4, IL-10 and IFN-alpha.
  • The assay functioned on a fluorescent bead-based system that allowed for the simultaneous detection of the cytokines in one single sample, achieved through a Luminex analyzer.
  • In order to quantify the individual cytokines, equine recombinant cytokine/IgG fusion proteins were used and validated as standards.

Performance of the Multiplex Assay

  • The analytical sensitivities of the multiplex assay were 40 pg/ml for IL-4 and 15 pg/ml for IL-10 and IFN-alpha.
  • When compared to the sensitivity of the previously used ELISA method, the new multiplex assay’s sensitivity increased by 13 to 150 times.
  • To validate the multiplex assay’s specificity, cell culture supernatants were used from equine peripheral blood mononuclear cells (PBMC) which were stimulated with different mitogens or infected with equine herpes virus type 1 (EHV-1).

Results and Further Potential

  • The detection pattern resulted in the multiplex analysis and assays were performed with individual standard cytokines.
  • It was found that individual bead assays did not interfere or cross-react during the simultaneous detection of equine IL-4, IL-10 and IFN-alpha.
  • This pioneered equine cytokine multiplex assay can be employed for various immunological applications and is notably cost-effective for the quantification of IL-4, IL-10 and IFN-alpha.
  • In the future, the assay has the potential to be expanded by adding bead assays for other equine cytokines and chemokines to the existing platform.

Cite This Article

APA
Wagner B, Freer H. (2008). Development of a bead-based multiplex assay for simultaneous quantification of cytokines in horses. Vet Immunol Immunopathol, 127(3-4), 242-248. https://doi.org/10.1016/j.vetimm.2008.10.313

Publication

Veterinary immunology and immunopathology
ISSN: 0165-2427
NlmUniqueID: 8002006
Country: Netherlands
Language: English
Volume: 127
Issue: 3-4
Pages: 242-248

Researcher Affiliations

Wagner, Bettina
  • Department of Population Medicine and Diagnostic Sciences, Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. bw73@cornell.edu
Freer, Heather

    MeSH Terms

    • Animals
    • Biological Assay / methods
    • Biological Assay / veterinary
    • Cytokines / metabolism
    • Enzyme-Linked Immunosorbent Assay / veterinary
    • Horses / metabolism
    • Immunoglobulin G

    Citations

    This article has been cited 40 times.
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