Simultaneous detection of porcine cytokines by multiplex analysis: development of magnetic bioplex assay

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.