Critical Care Research Group, The Prince Charles Hospital, Brisbane, Australia; Faculty of Medicine, University of Queensland, Brisbane, Australia.
Critical Care Research Group, The Prince Charles Hospital, Brisbane, Australia; Faculty of Medicine, University of Queensland, Brisbane, Australia; Research and Development, Australian Red Cross Lifeblood, Brisbane, Australia.
J Immunol Methods. 2020 Nov;486:112835. doi: 10.1016/j.jim.2020.112835. Epub 2020 Aug 20.
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.
越来越多的证据表明，炎症是许多常见疾病的基础。炎症/免疫调节/免疫介质，如细胞因子，是炎症的关键调节剂，介导免疫细胞募集和复杂的细胞内信号通路。在临床前研究中，羊模型越来越多地被应用，但现有的细胞因子水平测量方法存在局限性。我们建立并验证了羊血浆中白细胞介素（IL）-1β、IL-6、IL-8 和 IL-10 的酶联免疫吸附测定（ELISA）法。这些 ELISA 法具有高灵敏度和特异性，批内和批间 CV 低于 10%，回收率在 82%至 123%之间。IL-1β、IL-6、IL-8 和 IL-10 的灵敏度分别为 117.6 pg/mL、443.1 pg/mL、30.9 pg/mL 和 64.3 pg/mL。ELISA 测试结果在-80°C 下储存 12 周后，重现性显著下降。因此，为了准确测量细胞因子，需要在样本采集后三个月内测试血浆样本，以考虑细胞因子蛋白降解。这些 ELISA 法为鉴定羊的炎症性细胞因子变化提供了可靠和方便的方法，使我们能够深入了解这些反刍动物的疾病发病机制。
