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

J Immunol Methods. 2020 Nov;486:112835. doi: 10.1016/j.jim.2020.112835. Epub 2020 Aug 20.

Abstract

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.

Keywords: Cytokines; ELISA; Inflammation; Sheep.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Animals
  • Blood Specimen Collection
  • Cold Temperature
  • Enzyme-Linked Immunosorbent Assay*
  • Inflammation Mediators / blood*
  • Interleukin-10 / blood*
  • Interleukin-1beta / blood*
  • Interleukin-6 / blood*
  • Interleukin-8 / blood*
  • Protein Denaturation
  • Protein Stability
  • Reproducibility of Results
  • Sheep, Domestic
  • Time Factors

Substances

  • Inflammation Mediators
  • Interleukin-1beta
  • Interleukin-6
  • Interleukin-8
  • Interleukin-10