Wyns Heidi, Croubels Siska, Demeyere Kristel, Watteyn Anneleen, De Backer Patrick, Meyer Evelyne
Department of Pharmacology, Toxicology and Biochemistry, Ghent University, Faculty of Veterinary Medicine, Salisburylaan 133, 9820 Merelbeke, Belgium.
Vet Immunol Immunopathol. 2013 Jan 15;151(1-2):28-36. doi: 10.1016/j.vetimm.2012.09.041. Epub 2012 Oct 24.
Lipopolysaccharide (LPS) has been widely used as a model of immune challenge in pigs as it induces the immediate synthesis of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6, which trigger the production of the acute phase proteins (APPs) C-reactive protein (CRP), haptoglobin (Hp) and pig-Major Acute Phase Protein (pig-MAP). To measure secreted proteins in porcine plasma, specific and sensitive Enzyme-Linked Immuno Sorbent Assays (ELISAs) are well-suited to perform single parameter analysis, yet this approach is time-consuming and expensive for multi-parameter analyses. During the last decade, multiplex bead-based flow cytometry has been increasingly applied as it offers the opportunity to estimate protein ratios in a small sample volume. Cytometric bead array (CBA) is a flow cytometric application using a diversity of beads with unique fluorescence intensities, covalently coupled to a capture antibody for each protein of interest. Detection antibodies, either directly or indirectly conjugated to a fluorochrome, are added to accomplish the desired sandwich format. The aim of the present study was to develop a CBA 3-plex assay for the major pro-inflammatory cytokines TNF-α, IL-1β and IL-6, and an additional CBA 2-plex assay for the major APPs, CRP and pig-MAP, in porcine plasma. Results were compared to commercial ELISA kits. For the CBA 3-plex assay, the limits of detection (LODs) varied between 0.005 and 0.363 ng/mL, the intra- and inter-assay coefficients of variation were <10% and <16%, respectively. For TNF-α, IL-1β, IL-6 and pig-MAP, CBA time-concentration profiles similar to those obtained with commercial ELISAs were observed. In conclusion, the novel validated CBA 3-plex assay provides a fast and economical screening tool for determination of pro-inflammatory cytokine profiles in limited porcine plasma volumes. This tool will be applied to study the immunomodulatory properties of drugs in a porcine LPS inflammation model. This study also demonstrated the applicability of CBA for measurement of APPs in pigs, although a different combination than pig-MAP with CRP is recommended.
脂多糖（LPS）已被广泛用作猪免疫应激的模型，因为它能诱导促炎细胞因子如肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和IL-6的即时合成，这些细胞因子会触发急性期蛋白（APPs）C反应蛋白（CRP）、触珠蛋白（Hp）和猪主要急性期蛋白（pig-MAP）的产生。为了测定猪血浆中的分泌蛋白，特异性和灵敏的酶联免疫吸附测定（ELISA）非常适合进行单参数分析，但这种方法对于多参数分析来说既耗时又昂贵。在过去十年中，基于微珠的多重流式细胞术越来越多地被应用，因为它提供了在小样本体积中估计蛋白质比例的机会。细胞计数微珠阵列（CBA）是一种流式细胞术应用，使用具有独特荧光强度的多种微珠，这些微珠与针对每种目标蛋白质的捕获抗体共价偶联。添加直接或间接与荧光染料偶联的检测抗体以完成所需的夹心形式。本研究的目的是开发一种用于检测猪血浆中主要促炎细胞因子TNF-α、IL-1β和IL-6的CBA三联检测法，以及另一种用于检测主要APPs CRP和pig-MAP的CBA双联检测法。将结果与商业ELISA试剂盒进行比较。对于CBA三联检测法，检测限（LODs）在0.005至0.363 ng/mL之间，批内和批间变异系数分别<10%和<16%。对于TNF-α、IL-1β、IL-6和pig-MAP，观察到CBA时间-浓度曲线与使用商业ELISA获得的曲线相似。总之，新验证的CBA三联检测法为在有限的猪血浆体积中测定促炎细胞因子谱提供了一种快速且经济的筛选工具。该工具将用于研究猪LPS炎症模型中药物的免疫调节特性。本研究还证明了CBA在猪中测量APPs的适用性，尽管建议使用与pig-MAP和CRP不同的组合。
