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

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.