Di Nardo F, Anfossi L, Ozella L, Saccani A, Giovannoli C, Spano G, Baggiani C
Department of Chemistry, University of Turin, Via Giuria, 5, I-10125 Turin, Italy.
Department of Chemistry, University of Turin, Via Giuria, 5, I-10125 Turin, Italy.
J Chromatogr B Analyt Technol Biomed Life Sci. 2016 Aug 15;1028:192-198. doi: 10.1016/j.jchromb.2016.06.019. Epub 2016 Jun 16.
Salivary cortisol is regarded as a reliable parameter for the noninvasive assessment of the welfare of animals, because it is strictly related to stress levels. Several methods are available for salivary cortisol measurement in mammals, however rapid diagnostic test for detecting salivary cortisol are confined to humans. The availability of such non invasive diagnostic tools operable in situ would facilitate monitoring of animal welfare. The Cortisol stress™ test provides a simple and rapid tool to discriminate cortisol levels in canine saliva above or below 4ng/ml, which has been suggested as the cut-off value for distinguishing unstressed dogs from those experiencing stress. The test is based on a competitive immunochromatographic assay (ICT) using gold nanoparticles as probes, in which the color intensity of the Test line is inversely correlated to the salivary cortisol level. The qualitative result is obtained by the visual observation of the color formed on the Test line compared to that of the Control line We evaluated the accuracy of the test by determining salivary cortisol in 85 samples of canine saliva belonging to dogs with very variable age, sex, breed, and life history, and comparing the qualitative results to those obtained by a reference ELISA kit. Agreeing results were obtained through the two methods, and the ICT showed high diagnostic sensitivity, specificity and efficiency (100%, 98.4%, and 98.8%, respectively). Furthermore, we evaluated the precision of the test by an experimental design approach, which combines errors due to within-day and between-day variation with the biological variability, and demonstrated that the test could be reliably applied for correctly classifying canine samples, according to their salivary cortisol level. Moreover, we studied the shelf-life of the device in three experimental conditions. We confirmed the stability of the ICT at 4°C and 25°C for at least six months and observed similar results for an accelerated stability study conducted for 7days at 37°C, which suggest that the stability of ICT device could be estimated by the accelerated experiment alternatively to the real-time study.
唾液皮质醇被视为用于动物福利无创评估的可靠参数，因为它与应激水平密切相关。哺乳动物唾液皮质醇测量有多种方法，然而用于检测唾液皮质醇的快速诊断测试仅限于人类。此类可原位操作的无创诊断工具的出现将有助于监测动物福利。皮质醇应激™测试提供了一种简单快速的工具，可区分犬类唾液中皮质醇水平高于或低于4ng/ml，4ng/ml被建议作为区分无应激犬和有应激犬的临界值。该测试基于使用金纳米颗粒作为探针的竞争性免疫层析分析（ICT），其中测试线的颜色强度与唾液皮质醇水平呈负相关。通过目视观察测试线与对照线形成的颜色来获得定性结果。我们通过测定85份年龄、性别、品种和生活史差异很大的犬类唾液样本中的唾液皮质醇，并将定性结果与参考ELISA试剂盒获得的结果进行比较，评估了该测试的准确性。两种方法得到了一致的结果，ICT显示出高诊断敏感性、特异性和效率（分别为100%、98.4%和98.8%）。此外，我们通过实验设计方法评估了该测试的精密度，该方法将日内和日间变化引起的误差与生物变异性相结合，并证明该测试可根据犬类样本的唾液皮质醇水平可靠地用于正确分类。此外，我们在三种实验条件下研究了该设备的保质期。我们证实了ICT在4°C和25°C下至少六个月的稳定性，并在37°C下进行7天的加速稳定性研究中观察到类似结果，这表明ICT设备的稳定性可以通过加速实验而非实时研究来估计。
