Validation of an enzyme-linked immunosorbent assay (ELISA) for the measurement of canine S100A12. Academic Article uri icon

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.

published proceedings

  • Vet Clin Pathol

altmetric score

  • 0.25

author list (cited authors)

  • Heilmann, R. M., Cranford, S. M., Ambrus, A., Grützner, N., Schellenberg, S., Ruaux, C. G., Suchodolski, J. S., & Steiner, J. M.

citation count

  • 16

complete list of authors

  • Heilmann, Romy M||Cranford, Shannon M||Ambrus, Andy||Grützner, Niels||Schellenberg, Stefan||Ruaux, Craig G||Suchodolski, Jan S||Steiner, Jörg M

publication date

  • January 2016

publisher