Simultaneous detection of porcine proinflammatory cytokines using multiplex flow cytometry by the xMAP™ technology

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Abstract
Background: Multiplex flow cytometry is in widespread use for detection of cytokines in human samples. However, no report on the measurement of porcine cytokines using this method has previously been published. We report on the detection of the porcine proinflammatory cytokines TNF-α, IL-8, and IL-1β by the xMap-assay for multiplex flow cytometry. Methods: Commercially available antibodies to porcine cytokines were used as capture antibodies by attaching them to goat anti-mouse IgG coated microspheres with different fluorescent signatures. By the use of biotinylated detection antibodies and SAv-PE the amount of cytokines bound to the spheres were measured. Experiments were performed to determine the limits of detection and the amount of crossreactivity in buffer, serum, and plasma, using spiking with recombinant porcine cytokines. Results: The limit of detection ranged from 0.18 to 12 ng/ml. Generally, the detection limit was higher in serum and plasma, than in buffer. No crossreactivity between reagents was found. Conclusions: Porcine proinflammatory cytokines can be detected utilizing this method with satisfactory detection limits, and no crossreaction between the reagents involved. © 2006 International Society for Analytical Cytology