Dernfalk Johanna, Persson Waller Karin, Johannisson Anders
Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, P.O. Box 7011, SE-750 07 Uppsala, Sweden.
Vet Immunol Immunopathol. 2007 Jul 15;118(1-2):40-9. doi: 10.1016/j.vetimm.2007.04.004. Epub 2007 Apr 19.
Infectious diseases can cause large health problems in cattle. The infections cause an acute inflammatory response, mediated by pro-inflammatory cytokines such as IL-1beta, IL-6 and TNF-alpha. By mapping the pattern of cytokines during inflammations, valuable information about the course of an infection is gained. The aim of the present study was to evaluate a particle-based flow cytometric method, the xMAP technique, using ovine/bovine reagents, for quantification of IL-1beta, IL-6 and TNF-alpha, for application in studies on ruminant infectious diseases with emphasis on bovine milk and plasma samples. Singleplex, duplex and triplex xMAP assays were evaluated, and limits of detection (LODs) as well as intra- and inter-assay variabilities were determined for each assay. Cross-reactivity between reagents in multiplex assays was also tested. In addition, presence of cytokines in milk and plasma samples from healthy and mastitic cows was studied. The LODs were significantly lower for singleplex xMAP assays than for duplex and triplex assays. In singleplex assays, the LODs were 0.08, 0.2 and 0.5 ng/ml, for IL-1beta, IL-6 and TNF-alpha, respectively. Corresponding LODs in triplex assays were 2.0, 6.5 and 3.5 ng/ml. Data indicate that the linear ranges of the multiplex assays were narrower than in singleplex assays. The intra-assay coefficients of variation were < or =10.7% for singleplex assays, while they ranged from 6.2 to 23.2% in the triplex assay. The inter-assay variance ranged from 5.1 to 35.8% in singleplex assays, and from 8.8 to 78.4% in triplex assays. Cross-reactivity between reagents was not observed, and all three cytokines were detected in bovine milk and plasma samples collected from cows with clinical mastitis. In conclusion, our results show that the xMAP technique can be used for quantification of IL-1beta, IL-6 and TNF-alpha in bovine samples, and that further work is required to optimize the multiplex assays.
传染病会给牛群带来严重的健康问题。这些感染会引发急性炎症反应，由促炎细胞因子如白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）介导。通过绘制炎症过程中细胞因子的模式，可以获得有关感染进程的有价值信息。本研究的目的是评估一种基于颗粒的流式细胞术方法——xMAP技术，使用绵羊/牛试剂来定量IL-1β、IL-6和TNF-α，用于反刍动物传染病研究，重点是牛奶和血浆样本。评估了单重、双重和三重xMAP检测方法，并确定了每种检测方法的检测限（LOD）以及批内和批间变异性。还测试了多重检测中试剂之间的交叉反应性。此外，研究了健康奶牛和患乳腺炎奶牛的牛奶和血浆样本中细胞因子的存在情况。单重xMAP检测方法的检测限显著低于双重和三重检测方法。在单重检测中，IL-1β、IL-6和TNF-α的检测限分别为0.08、0.2和0.5 ng/ml。三重检测中的相应检测限为2.0、6.5和3.5 ng/ml。数据表明，多重检测的线性范围比单重检测窄。单重检测的批内变异系数≤10.7%，而三重检测的变异系数为6.2%至23.2%。单重检测的批间方差为5.1%至35.8%，三重检测的批间方差为8.8%至78.4%。未观察到试剂之间的交叉反应性，并且在从临床乳腺炎奶牛采集的牛奶和血浆样本中检测到了所有三种细胞因子。总之，我们的结果表明，xMAP技术可用于定量牛样本中的IL-1β、IL-6和TNF-α，并且需要进一步开展工作来优化多重检测方法。
