title: Validation of an enzyme-linked immunosorbent assay (ELISA) for the measurement of canine S100A12.
abstract: BACKGROUND
  Canine S100 calcium-binding protein A12 (cS100A12) shows promise as biomarker of inflammation in dogs. A previously developed cS100A12-radioimmunoassay (RIA) requires radioactive tracers and is not sensitive enough for fecal cS100A12 concentrations in 79% of tested healthy dogs. An ELISA assay may be more sensitive than RIA and does not require radioactive tracers.
  
  
  OBJECTIVE
  The purpose of the study was to establish a sandwich ELISA for serum and fecal cS100A12, and to establish reference intervals (RI) for normal healthy canine serum and feces.
  
  
  METHODS
  Polyclonal rabbit anti-cS100A12 antibodies were generated and tested by Western blotting and immunohistochemistry. A sandwich ELISA was developed and validated, including accuracy and precision, and agreement with cS100A12-RIA. The RI, stability, and biologic variation in fecal cS100A12, and the effect of corticosteroids on serum cS100A12 were evaluated.
  
  
  RESULTS
  Lower detection limits were 5 μg/L (serum) and 1 ng/g (fecal), respectively. Intra- and inter-assay coefficients of variation were ≤ 4.4% and ≤ 10.9%, respectively. Observed-to-expected ratios for linearity and spiking recovery were 98.2 ± 9.8% (mean ± SD) and 93.0 ± 6.1%, respectively. There was a significant bias between the ELISA and the RIA. The RI was 49-320 μg/L for serum and 2-484 ng/g for fecal cS100A12. Fecal cS100A12 was stable for 7 days at 23, 4, -20, and -80°C; biologic variation was negligible but variation within one fecal sample was significant. Corticosteroid treatment had no clinically significant effect on serum cS100A12 concentrations.
  
  
  CONCLUSIONS
  The cS100A12-ELISA is a precise and accurate assay for serum and fecal cS100A12 in dogs.
authors.affiliation: Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Romy M
authors.initials: RM
authors.lastname: Heilmann
authors.affiliation: Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Shannon M
authors.initials: SM
authors.lastname: Cranford
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Andy
authors.initials: A
authors.lastname: Ambrus
authors.affiliation: Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Niels
authors.initials: N
authors.lastname: Grützner
authors.affiliation: Clinic for Small Animal Internal Medicine, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schellenberg
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR, USA.
authors.forename: Craig G
authors.initials: CG
authors.lastname: Ruaux
authors.affiliation: Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Jan S
authors.initials: JS
authors.lastname: Suchodolski
authors.affiliation: Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: Jörg M
authors.initials: JM
authors.lastname: Steiner
mesh_terms: D000818:Animals; D004283:Dog Diseases / Q000097:blood*; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary*; D005243:Feces / Q000737:chemistry; D005260:Female; D008297:Male; D011817:Rabbits; D012016:Reference Values; D015203:Reproducibility of Results; D000067616:S100A12 Protein / Q000032:analysis* / Q000097:blood
chemical_list: D000067616:S100A12 Protein
keywords: Calgranulin; RIA; immunohistochemistry; inflammation; stability