Multiplex flow cytometric assays for markers of inflammation

The aim of this thesis was to develop new techniques for quantification of bovine pro-inflammatory markers, with emphasis on cytokines and acute phase proteins, and to apply the techniques, using mastitis as disease model. Singleplex, duplex and triplex xMAP assays were developed for the bovine cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1β.(IL-1beta), interleukin-6 (IL-6), and the bovine acute phase proteins serum amyloid A (SAA) and lipopolysaccharide binding protein (LBP). Detection limits, linear ranges and intra- and inter-assay variations differed between assays, but generally, lower detection limits and wider linear ranges were observed in singleplex assays than in duplex and triplex assays. The detection limits for TNF-alpha, IL-1beta and IL-6 were satisfactory in the singleplex assays. Further development work is required before the multiplex formats can be used in assays where very low cytokine concentrations are of interest. In the assays for acute phase proteins the detection limits and linear ranges were satisfactory, but could probably be further improved. All xMAP assays could be used for quantification of the analytes in milk and plasma samples from cows with experimentally induced Escherichia coli or Staphylococcus aureus mastitis. Simultaneous detection of IL-1β, SAA and LBP was performed in plasma. LBP is secreted solely during bacterial infections, and cytokines and acute phase proteins are secreted at different phases of the inflammation. Thus a time perspective and information whether the infection is of bacterial origin or not is provided by measuring IL-1β, SAA and LBP simultaneously. In one of the studies, ten cows were grouped as high or low responders for TNF-α, IL-1β and IL-6, based on their cytokine response in an ex vivo whole blood stimulation assay (WBA) with lipopolysaccharide (LPS) and E. coli. After the WBA, the cows were intramammary inoculated with E. coli, and the WBA was evaluated for its usefulness as a predictive tool of the severity of E. coli mastitis. The pre-inoculation WBA with used stimulation doses and incubation times could not predict the severity of an E. coli induced mastitis