Detection of bovine inflammatory cytokines IL-1ß, IL-6, and TNF-a with a multiplex electrochemiluminescent assay platform

Authors: Chitko-Mckown, Carol; Bierman, Stacy; Kuehn, Larry; Bennett, Gary; DEDONDER, KEITH - Kansas State University; APLEY, MICHAEL - Kansas State University; Harhay, Gregory; Clawson, Michael - Mike; WHITE, BRADLEY - Kansas State University; LARSON, ROBERT - Kansas State University; CAPIK, SARAH - Texas A&M Agrilife; LUBBERS, BRIAN - Kansas State University

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/20/2021
Publication Date: 7/1/2021
Publication URL: https://handle.nal.usda.gov/10113/7391751
Citation: Chitko-McKown, C.G., Bierman, S.L., Kuehn, L.A., Bennett, G.L., DeDonder, K.D., Apley, M.D., Harhay, G.P., Clawson, M.L., White, B.J., Larson, R.L., Capik, S.F., Lubbers, B.V. 2021. Detection of bovine inflammatory cytokines IL-1ß, IL-6, and TNF-a with a multiplex electrochemiluminescent assay platform. Veterinary Immunology and Immunopathology. 237. Article 110274. https://doi.org/10.1016/j.vetimm.2021.110274.
DOI: https://doi.org/10.1016/j.vetimm.2021.110274

Interpretive Summary: Multiplex assays are those which can measure more than a single metabolite at a time. By running multiple assays concurrently, less sample can be used and time and variation can also be reduced. Unfortunately, there are few commercially available multiplex assays for bovine cytokines - important proteins that are part of the immune system. We purchased "Do-It-Yourself" ELISA kits that are marketed to perform individual assays for the bovine inflammatory cytokines IL-1beta, IL-6 and TNF-alpha and adapted them for use in the multiplex Meso Scale Discovery U-PLEX platform. 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. In our hands the U-PLEX platform was a viable means to develop analyte- and species-specific multiplex assays using privately developed or purchased sets of commercially available reagents.

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 standard, were purchased for each of these three cytokines. The biotinylated antibodies 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 – 50,000 pg/ml, and for IL-6 from 0 – 10,000 pg/ml. The average lowest level of detection concentration measured by the standard curves were 5.3 pg/ml, 0.92 pg/ml, and 22.34 pg/ml for IL-1ß, IL-6, and TNF-a respectively, as determined by data from seven plates containing bovine plasma samples from a combination of healthy and diseased cattle. The U-PLEX platform was a viable means to develop custom analyte- and species-specific multiplex assays using privately developed or purchased sets of commercially available reagents.