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

Heilmann, Romy M; Cranford, Shannon M; Ambrus, Andy; Grützner, Niels; Schellenberg, Stefan; Ruaux, Craig G; Suchodolski, Jan S; Steiner, Jörg M (2016). Validation of an enzyme-linked immunosorbent assay (ELISA) for the measurement of canine S100A12. Veterinary clinical pathology, 45(1), pp. 135-147. Wiley-Blackwell 10.1111/vcp.12320

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

05 Veterinary Medicine > Department of Clinical Veterinary Medicine (DKV) > Swine Clinic
05 Veterinary Medicine > Department of Clinical Veterinary Medicine (DKV)

UniBE Contributor:

Grützner, Niels

Subjects:

600 Technology > 610 Medicine & health

ISSN:

0275-6382

Publisher:

Wiley-Blackwell

Language:

English

Submitter:

Daniela Fasel

Date Deposited:

09 Jun 2016 12:40

Last Modified:

10 Sep 2017 18:18

Publisher DOI:

10.1111/vcp.12320

PubMed ID:

26765807

Uncontrolled Keywords:

Calgranulin, RIA, immunohistochemistry, inflammation, stability

BORIS DOI:

10.7892/boris.81382

URI:

https://boris.unibe.ch/id/eprint/81382

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