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