Evaluation of the suitability of monoclonal antibodies for flow cytometric intracellular cytokine detection in porcine peripheral blood lymphocytes

摘要

Panels of monoclonal antibodies (mAbs) specific for porcine interleukin (IL)-2, IL-4, IL-6, IL-12, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha were evaluated for their applicability in flow cytometric intracellular cytokine detection. Peripheral blood mononuclear cells were short-time stimulated in the presence of brefeldin-A, ionomycin and phorbol-12-myristate-13-acetate, fixed and incubated with the respective mAbs as well as phycoerythrin-conjugated second-step antibodies. Suitability of mAbs was judged by use of statistical data and by visual control of scattergrams, considering the capability of mAbs to discriminate between cytokine-positive and cytokine-negative cell populations. The number of suitable clones differed to a high degree between the investigated cytokines, but at least one mAb fitting for flow cytometric intracellular cytokine detection could be identified within each panel. Monoclonal Abs producing scattergrams with a clear demarcation between positive and negative cell populations were found within the anti-IL-2, IL-6 and IFN-gamma panels, whereas less well defined positive and negative cell populations could be generated by use of mAbs within the anti-IL-4 and TNF-alpha panels. Only one moderately fitting mAb was identified within the anti-IL-12 panel. After having evaluated the best fitting mAbs, these were used to obtain reference levels for the physiological range of porcine lymphocytic cytokine production in a second set of experiments. For that reason, 13 clinically healthy pigs aged between 6 weeks and 6 months were investigated. Data presented are given as mean +/- SD of the percentage of positive-staining lymphocytes: IL-2, 45.5 +/- 27.6; IL-4, 34.1 +/- 21.3; IL-6, 45.4 +/- 23.8; IL-12, 13.9 +/- 8.6; TNF-alpha, 43.4 +/- 11.3; IFN-gamma, 65.5 +/- 14.8.