Optimization of a bovine cytokine multiplex assay using a new bovine and cross-reactive equine monoclonal antibodies.

Cytokines are important markers for immune activation, regulation, and homeostasis. The lack of monoclonal antibodies (mAbs) and sensitive assays to evaluate cytokine secretion has hindered research of bovine inflammation and immune regulation. We recently developed a fluorescent bead-based multiplex assay (multiplex assay) for bovine IL-10, TNF-α, and IFN-γ. Although the original assay covers a broad concentration range for the 3 targets, analytical sensitivity for IL-10 and IFN-γ could be improved to facilitate detection of these cytokines in their physiological low pg/mL range. To optimize the multiplex assay, we generated a new bovine IL-10 mAb and explored its use for the detection of intracellular and secreted bovine IL-10. The new bovine IL-10 mAb 130 recognized recombinant bovine IL-10 fusion protein and did not react with the fusion protein tag, or the TNF-α and IFN-γ standards in the multiplex assay. For improving IFN-γ detection, we explored cross-reactivity of anti-equine IFN-γ mAbs by intracellular staining of bovine stimulated peripheral blood mononuclear cells (PBMC). Equine IFN-γ mAb 3 showed excellent cross-reactivity with bovine IFN-γ by intracellular detection. Adding IL-10 mAb 130 and IFN-γ mAb 3 to the bovine multiplex assay substantially improved the analytical sensitivity with lower limits of detection in the low pg/mL range for all analytes. The detection ranges for the optimized multiplex assay were determined as 2 - 134,000 pg/mL for IL-10, 8 - 127,000 pg/mL for IFN-γ, and 12 - 193,000 pg/mL for TNF-α. The assay was next used to measure cytokine concentrations in cell culture supernatants from PBMC stimulated in plasma from whole blood stimulation to confirm native IL-10, TNF-α, and IFN-γ recognition and to explore the upper detection limits of the assay. In PBMC stimulation with a mix of phorbol myristate acetate (PMA) and ionomycin resulted in highest cytokine concentrations, while in plasma from whole blood stimulation, highest concentrations were observed in samples stimulated with a mix of lipopolysaccharide (LPS), phytohemagglutinin (PHA), and the TLR-2/6 agonist Pam2Csk4. PBMC and whole blood stimulation protocols showed that the optimized multiplex assay covers a wide linear detection range for measuring cytokine concentrations in bovine samples. For whole blood stimulation, a cocktail of pathogen associated molecular patterns elicited a stronger cytokine response than a mix of PMA and ionomycin, but response varied considerably between individual cattle. In conclusion, optimizing the bovine cytokine assay with new reagents improved the lower detection limits and widened the linear detection ranges while lowering the background of the multiplex assay.