Sheep are used to model clinical conditions and interventions. To increase the validity of these models and facilitate investigation of the underlying mechanisms of pathological changes, an understanding of the inflammatory changes would be beneficial. In-house assays can be problematic to reproduce in other laboratories, and can be dependent upon reagents not commercially available. We therefore aimed to evaluate several commercial assays that quantify sheep cytokines.
Blood from a Merino ewe was stimulated with lipopolysaccharide (LPS; 100 ng/mL, 6hrs, 37°C) and plasma was harvested and stored (-80°C). Dilutions of this plasma and sheep cytokines (Cusabio: IL-1β, IL-6, IL-10 and TNF-α; Kingfisher Biotech: IL-1β, IL-6 and TNF-α; Moredun Research Institute: IL-1β, IL-6, IL-10 and TNF-α) were assayed using ELISA kits from Cusabio according to instructions.
Standard curves for IL-1β, IL-6, IL-10 and TNF-α ELISA kits performed well (r2 = 0.9911, 0.9948, 0.9978 and 0.9881 respectively). These kits did not quantify the recombinant sheep cytokines assayed (ODs were below the standard curve or calculated concentrations did not correspond to concentrations tested). The kits may have been capable of detecting native cytokines present in the plasma sample (IL-1β: undiluted = 19.2pg/mL; 1/2 diluted = 6.3pg/mL; 1/4 diluted undetectable; IL-6: 1/2 diluted = 22.8pg/mL, 1/4 diluted = 33.6pg/mL; IL-10: undiluted = 12.3pg/mL; 1/2 diluted = 18.7pg/mL, 1/4 diluted =30.8pg/mL; and TNF-α: undiluted = 10.1pg/mL, 1/2 diluted < 6.25pg/mL); however, the levels were lower than those expected following similar LPS stimulation of human blood.
The Cusabio ELISA kits tested here failed to quantify any of the panel of recombinant ovine cytokines tested, but may have been capable of quantifying native protein expected to be present in the plasma from LPS stimulated sheep’s blood. Further studies using established methods are needed to quantify cytokine levels in this plasma sample.