Chemiluminescent assay as an alternative to radioimmunoassay for the measurement of cortisol in plasma and skin mucus of Oncorhynchus mykiss

Abstract

The aims of the present study were to validate an automated chemiluminescence assay (CIA) for cortisol determination in plasma and skin mucus of fish, to compare the results produced with those obtained by radioimmunoassay (RIA), and to evaluate the assay capacity to differentiate between stressed and non-stressed fish.Cortisol hormone was measured using both CIA and RIA in plasma and skin mucus of 36 rainbow trout (Oncorhynchus mykiss) at different time points after 3 min of air exposure. For CIA, analytical validation consisting of intra- and inter-assay coefficient of variation (CV), limit of detection, and linearity studies were performed. In addition, correlation and agreement between the CIA and RIA were evaluated.In all cases, intra- and inter-assay CV for CIA measurements were lower than 10 and 11%, respectively. Cortisol results with CIA were statistically significantly higher than those obtained with RIA in both tissues (p < 0.001). Strong positive correlation was observed between the two methods (r = 0.999, p < 0.001 and r = 0.993, p = 0.03 for plasma and skin mucus, respectively). The agreement between the two techniques was examined by Bland-Altman plots, which identified wide confidence intervals and outliers for cortisol (plasma n = 3, skin mucus n = 2) results. Both assays were able to differentiate between pre- and post-stressed fish in both biological matrices.In conclusion, CIA assay is precise and accurate for measuring cortisol in plasma and skin mucus of fish and is able to discriminate among stressed and control animals, showing a strong correlation with RIA, making it a reliable method for stress assessment in fish.