Location: Animal Health GenomicsTitle: Development of a multiplex assay for the bovine inflammatory cytokines IL-1ß, IL-6, and TNF-a
Submitted to: Immunology
Publication Type: Abstract Only
Publication Acceptance Date: 2/5/2020
Publication Date: 5/1/2020
Citation: Chitko-McKown, C.G., Bierman, S.L. 2020. Development of a multiplex assay for the bovine inflammatory cytokines IL-1ß, IL-6, and TNF-a [abstract]. Immunology. 204 (Supplement 1):92.41.
Technical Abstract: Commercially available bovine-specific assays are limited in number, and multiplex assays for this species are rare. Our objective was to develop a multiplex assay for the bovine inflammatory cytokines IL-1ß, IL-6, and TNF-a using the Meso Scale Discovery U-PLEX platform. "Do-It-Yourself" ELISA kits that contained polyclonal antibodies, both unlabeled and biotinylated, and the specific recombinant bovine cytokine, were purchased for each of these three cytokines. The biotinylated antibodies that would normally be used as reporters in a traditional ELISA were coupled to linkers that bind to specific locations within each well of the U-PLEX plate. Unique linkers were used for each of the cytokines. The unlabeled antibodies were conjugated with electrochemiluminescent labels to serve as detection antibodies. Each cytokine assay was optimized individually prior to performing an optimization on the multiplex assay containing reagents for all three cytokines. To calculate cytokine concentrations, standard curves were developed using the recombinant cytokines and were run concurrently on each plate. Standard curves for IL-1ß and TNF-a were run at concentrations ranging from 0 - 50,000pg/ml, and for IL-6 from 0 - 10,000pg/ml. The minimum average concentrations measured by the standard curves were 5.3pg/ml, 0.92pg/ml, and 22.34pg/ml for IL-1ß, IL-6, and TNF-a respectively as determined by data from seven plates. Only 37 out of 280 unique samples assayed had IL-ß or TNF-a concentrations above the upper limits of these standard curves.