title: Critical appraisal of four IL-6 immunoassays.
abstract: BACKGROUND
  Interleukin-6 (IL-6) contributes to numerous inflammatory, metabolic, and physiologic pathways of disease. We evaluated four IL-6 immunoassays in order to identify a reliable assay for studies of metabolic and physical function. Serial plasma samples from intravenous glucose tolerance tests (IVGTTs), with expected rises in IL-6 concentrations, were used to test the face validity of the various assays.
  
  
  RESULTS
  IVGTTs, administered to 14 subjects, were performed with a single infusion of glucose (0.3 g/kg body mass) at time zero, a single infusion of insulin (0.025 U/kg body mass) at 20 minutes, and frequent blood collection from time zero to 180 minutes for subsequent Il-6 measurement. The performance metrics of four IL-6 detection methods were compared: Meso Scale Discovery immunoassay (MSD), an Invitrogen Luminex bead-based multiplex panel (LX), an Invitrogen Ultrasensitive Luminex bead-based singleplex assay (ULX), and R&D High Sensitivity ELISA (R&D). IL-6 concentrations measured with MSD, R&D and ULX correlated with each other (Pearson Correlation Coefficients r = 0.47-0.94, p<0.0001) but only ULX correlated (r = 0.31, p = 0.0027) with Invitrogen Luminex. MSD, R&D, and ULX, but not LX, detected increases in IL-6 in response to glucose. All plasma samples were measurable by MSD, while 35%, 1%, and 4.3% of samples were out of range when measured by LX, ULX, and R&D, respectively. Based on representative data from the MSD assay, baseline plasma IL-6 (0.90 ± 0.48 pg/mL) increased significantly as expected by 90 minutes (1.29 ± 0.59 pg/mL, p = 0.049), and continued rising through 3 hours (4.25 ± 3.67 pg/mL, p = 0.0048).
  
  
  CONCLUSIONS
  This study established the face validity of IL-6 measurement by MSD, R&D, and ULX but not LX, and the superiority of MSD with respect to dynamic range. Plasma IL-6 concentrations increase in response to glucose and insulin, consistent with both an early glucose-dependent response (detectable at 1-2 hours) and a late insulin-dependent response (detectable after 2 hours).
authors.affiliation: Division of Rheumatology, Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America. vbk@duke.edu
authors.forename: Dana K
authors.initials: DK
authors.lastname: Thompson
authors.forename: Kim M
authors.initials: KM
authors.lastname: Huffman
authors.forename: William E
authors.initials: WE
authors.lastname: Kraus
authors.forename: Virginia Byers
authors.initials: VB
authors.lastname: Kraus
mesh_terms: D005947:Glucose / Q000008:administration & dosage / Q000494:pharmacology; D006801:Humans; D007118:Immunoassay / Q000592:standards*; D007328:Insulin / Q000008:administration & dosage / Q000494:pharmacology; D015850:Interleukin-6 / Q000097:blood*; D007700:Kinetics; D013997:Time Factors
chemical_list: C508600:IL6 protein, human; D007328:Insulin; D015850:Interleukin-6; D005947:Glucose