Heilmann Romy M, Cranford Shannon M, Ambrus Andy, Grützner Niels, Schellenberg Stefan, Ruaux Craig G, Suchodolski Jan S, Steiner Jörg M
Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
Vet Clin Pathol. 2016 Mar;45(1):135-47. doi: 10.1111/vcp.12320. Epub 2016 Jan 14.
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.
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.
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.
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.
The cS100A12-ELISA is a precise and accurate assay for serum and fecal cS100A12 in dogs.
犬S100钙结合蛋白A12（cS100A12）有望成为犬炎症的生物标志物。先前开发的cS100A12放射免疫分析（RIA）需要放射性示踪剂，并且对79%的受试健康犬粪便中的cS100A12浓度不够敏感。酶联免疫吸附测定（ELISA）可能比RIA更敏感，且不需要放射性示踪剂。
本研究旨在建立一种用于血清和粪便cS100A12的夹心ELISA，并确定正常健康犬血清和粪便的参考区间（RI）。
制备多克隆兔抗cS100A12抗体，并通过蛋白质印迹法和免疫组织化学法进行检测。开发并验证了一种夹心ELISA，包括准确性和精密度，以及与cS100A12-RIA的一致性。评估了粪便cS100A12的RI、稳定性和生物学变异，以及皮质类固醇对血清cS100A12的影响。
检测下限分别为5μg/L（血清）和1ng/g（粪便）。批内和批间变异系数分别≤4.4%和≤10.9%。线性和加样回收率的观察值与预期值之比分别为98.2±9.8%（平均值±标准差）和93.0±6.1%。ELISA和RIA之间存在显著偏差。血清cS100A12的RI为49 - 320μg/L，粪便cS100A12的RI为2 - 484ng/g。粪便cS100A12在23、4、-20和-80°C下可稳定保存7天；生物学变异可忽略不计，但单个粪便样本内的变异显著。皮质类固醇治疗对血清cS100A12浓度无临床显著影响。
cS100A12-ELISA是一种用于检测犬血清和粪便中cS100A12的精确且准确的检测方法。
