Development and validation of ELISAs for the quantitation of interleukin (IL)-1β, IL-6, IL-8 and IL-10 in ovine plasma

• DOI: 10.1016/j.jim.2020.112835
• Publication Date: 2020-08-20T06:33:06Z
• Authors: Mahé Bouquet; Margaret R. Passmore; Louise E. See Hoe; John-Paul Tung; Gabriela Simonova; Ai-Ching Boon; John F. Fraser
• Keywords: Protein Denaturation; Time Factors; Immunology; Interleukin-1beta; 610; Enzyme-Linked Immunosorbent Assay; 03 medical and health sciences; Animals; Sheep, Domestic; Inflammation; 2403 Immunology; Blood Specimen Collection; 0303 health sciences; Sheep; Interleukin-6; Protein Stability; Interleukin-8; Reproducibility of Results; Ophthalmology and optometry; Medical microbiology; Interleukin-10; 3. Good health; Cold Temperature; 2723 Immunology and Allergy; Cytokines; ELISA; Inflammation Mediators

There is growing evidence that inflammation underpins many common diseases. Inflammatory/immunomodulatory/immune mediators, such as cytokines, are key modulators of inflammation and mediate both immune cell recruitment and complex intracellular signalling pathways. Ovine models of disease are increasingly utilized in pre-clinical research, however existing methods for measuring cytokine levels are limited. We established and validated enzyme-linked immunosorbent assays (ELISAs) targeting interleukin (IL)-1β, IL-6, IL-8 and IL-10 in sheep plasma. These ELISAs showed high sensitivity and specificity with intra- and inter-assay CV's below 10%, and recovery rates between 82 and 123%. Sensitivity for IL-1β, IL-6, IL-8 and IL-10 were 117.6 pg/mL, 443.1 pg/mL, 30.9 pg/mL, and 64.3 pg/mL, respectively. ELISA test result reproducibility decreased significantly after 12 weeks of plasma storage at -80 °C. Therefore, for accurate cytokine measurements, plasma samples need to be tested within three months of sample collection to account for cytokine protein degradation. These ELISAs offer a reliable and convenient method to identify inflammatory cytokine changes in sheep, allowing key insights into the disease pathogenesis of these ruminants.