title: Assessment of urine solute and matrix effects on the performance of an enzyme-linked immunosorbent assay for measurement of interleukin-6 in dog urine.
abstract: Measurement of cytokine concentrations within body fluids is a means of recognizing subclinical and/or unresolved, infectious and inflammatory states in patients. In the urinary tract, such information may be useful for identifying patients with pyelonephritis, asymptomatic bacteriuria, recurrent infections, and cystitis. One such cytokine, interleukin-6 (IL-6), is recognized as a primary cytokine that is produced following exposure of the urothelium to bacterial virulence factors. Complicating reliable testing for this and other cytokines is the nature of urine itself. Urine varies widely in its composition as indicated by the range of pH and urine specific gravity (USG) observed in healthy patients. An additional variable is the protein and carbohydrate matrix capable of hindering immunologic testing modalities, such as enzyme-linked immunosorbent assays (ELISAs). The purpose of the current study was to examine the role of urine pH, USG, and matrix while optimizing a canine-specific chemiluminescent ELISA for the measurement of IL-6 in the urine of dogs. Urine spiked with IL-6 obtained maximal IL-6 quantitative recoveries of only 55 ± 10% (mean ± 1 standard deviation) when an ELISA optimized for cell culture supernatants was used. The urine matrix and variations in USG were determined to by contributing to this poor IL-6 recovery. Using specific matrix inhibitors and optimal dilutions improved the IL-6 quantitative recovery to 91 ± 5%. Urine pH (5.5-9.5) had no effect. The current work underscores the importance of critically optimizing testing modalities for biomarkers, particularly if they are immunologic in origin.
authors.affiliation: College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA. michael_wood@ncsu.edu
authors.forename: Michael W
authors.initials: MW
authors.lastname: Wood
authors.forename: Sushila K
authors.initials: SK
authors.lastname: Nordone
authors.forename: Shelly L
authors.initials: SL
authors.lastname: Vaden
authors.forename: Edward B
authors.initials: EB
authors.lastname: Breitschwerdt
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000652:urine; D004283:Dog Diseases / Q000276:immunology* / Q000652:urine; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D006863:Hydrogen-Ion Concentration; D015850:Interleukin-6 / Q000652:urine*; D057230:Limit of Detection; D013046:Specific Gravity; D014552:Urinary Tract Infections / Q000276:immunology* / Q000652:urine
chemical_list: D015415:Biomarkers; D015850:Interleukin-6