Bjerre Mette, Hansen Troels Krarup, Flyvbjerg Allan, Tønnesen Else
Immunoendocrine Research Unit, The Medical Research Laboratories, Aarhus University Hospital, Aarhus C, DK-8000, Denmark.
Vet Immunol Immunopathol. 2009 Jul 15;130(1-2):53-8. doi: 10.1016/j.vetimm.2009.01.007. Epub 2009 Feb 23.
Multiplex assays for analysis of human and rodent cytokines are highly developed, while development of porcine cytokine assays needs further attention. In order to follow the cytokine response in consecutive porcine samples, in which the sample volume may be limited, we have developed multiplex immunoassays for simultaneous detection of porcine cytokines interleukin (IL)-1beta, IL-6, IL-8, IL-10, tumour necrosis factor alpha (TNF-alpha), and heat shock protein 32 (Hsp32). Antibodies against porcine cytokines were coupled to magnetic microspheres. Quantification was obtained with biotinylated antibodies followed by PE-labelled streptavidin and measurements by Luminex(100). Validation and cross-reaction experiments revealed detection limits below 5-20 ng/L, recovery of recombinant cytokines in spiked plasma between 80 and 110%, and intra- and inter-assay variation between 5 and 15%. No cross-reaction between assays was found. However, for optimal sensitivity the assays were performed as a 2-plex (IL-1beta and Hsp32) and a 4-plex (IL-6, IL-8, IL-10, and TNF-alpha). Cytokine levels were determined in plasma samples from a porcine model of acute endotoxaemia and the levels correlated to previously published concentrations.
用于分析人和啮齿动物细胞因子的多重检测方法已经高度发达，而猪细胞因子检测方法的开发则需要进一步关注。为了跟踪连续猪样本中的细胞因子反应（这些样本的体积可能有限），我们开发了多重免疫检测方法，用于同时检测猪细胞因子白细胞介素（IL）-1β、IL-6、IL-8、IL-10、肿瘤坏死因子α（TNF-α）和热休克蛋白32（Hsp32）。针对猪细胞因子的抗体与磁性微球偶联。通过生物素化抗体，然后用PE标记的链霉亲和素进行定量，并通过Luminex（100）进行测量。验证和交叉反应实验显示检测限低于5-20 ng/L，加标血浆中重组细胞因子的回收率在80%至110%之间，批内和批间变异在5%至15%之间。未发现检测方法之间的交叉反应。然而，为了获得最佳灵敏度，检测方法以2联检（IL-1β和Hsp32）和4联检（IL-6、IL-8、IL-10和TNF-α）的形式进行。在急性内毒素血症猪模型的血浆样本中测定了细胞因子水平，这些水平与先前发表的浓度相关。
