7 Validation of a commercial ELISA kit for non-invasive measurement of equine cortisol concentration

Abstract

The measurement of fecal corticosteroid metabolites (FCM) is a method for quantifying potential stress. The sampling method is relatively non-invasive, especially when compared with blood cortisol. Fecal collection reduces concern for a rise in cortisol from the sampling method itself and has been shown to be a more consistent measure of cortisol in horses without the daily diurnal rhythm observed in blood cortisol. Commercial ELISA kits offer many benefits over previously validated RIA methods but lack validation. Further, time point of fecal collection has not been established. The objective of this study was to provide both analytical and biologic validation of fecal and plasma samples from horses using a commercial cortisol ELISA Kit (Arbor Assays DetectX Cortisol ELISA kit, K003-H1). In October, horses (4 M, 4 F, 8 ± 7 yr) were transported in pairs for a 15-min transportation session using a livestock trailer that included a divider to prevent physical contact and limit visual contact. Blood samples were collected via jugular venipuncture before, immediately after, and 60 min post-transportation. Fecal samples were collected via drop or grab sampling before transportation and 20, 24, and 28 h post-transportation. Data were analyzed using PROC MIXED in SAS. Inter-assay and intra-assay coefficients of variation for FCM were 9.0 and 10.8%, respectively and for plasma cortisol 11.0 and 7.2%, respectively. Dilutionalparallelism was observed between dilutions 1:20 and 1:40 for fecal samples and 1:100 and 1:250 for plasma samples. The recovery of a known spike for plasma cortisol and FCM were 98.2 and 100.0%, respectively. Plasma cortisol concentrations increased in response to trailering (73.4 ± 7.6 ng/ml, 0 min post-transportation, P = 0.02) compared with pre-transportation (41.2 ± 7.6 ng/ml) and began to decline 60 min post-transportation (51.0 ± 7.6 ng/ml) when compared with immediately post-trailering (P = 0.09). Plasma cortisol concentrations pre-trailering were not different from 60 min post-trailering. FCM concentrations increased 24 h post-trailering (10.8 ± 1.7 ng/g, P = 0.04) when compared with pre-transportation (7.4 ± 1.7 ng/g). FCM concentrations pre-trailering were not different from 20 h (8.68 ± 1.7 ng/g, P = 0.64) or 28 h post-trailering (10.0 ± 1.7 ng/g, P = 0.11). These data support that 15 min of transport with limited visual/physical contact between conspecifics is adequate to elicit a stress response in horses and that changes in FCM are observed 24 h post-stressor. In conclusion, the Arbor Assay Cortisol ELISA kit is a reliable, economic option for the measurement of cortisol in equine plasma and fecal samples.