Validation of a microtitre plate ELISA for measuring cortisol in fish and comparison of stress responses of rainbow trout ( Oncorhynchus mykiss) and lake trout ( Salvelinus namaycush)

Abstract

A microtitre plate enzyme-linked immunoassay (ELISA) was validated for measuring serum cortisol in rainbow trout ( Oncorhynchus mykiss) and lake trout ( Salvelinus namaycush). The ELISA uses a highly specific rabbit antibody raised against cortisol-3-carboxymethyloxime (CMO)/bovine serum albumin (BSA), and a conjugate of horseradish peroxidase (HRP) also linked to cortisol in the 3-position by a CMO bridge. The chromatogenic substrate is 3,3′,5,5′-tetramethylbenzidine (TMB). The standard curve was linear (logit/log) from the lower limit of sensitivity of the assay (2 pg/well = approx. 100 pg/ml serum) to approximately 1000 pg/well. The ELISA satisfied the strictest criteria of specificity, reproducibility (interassay coefficient of variation < 11%), precision (intraassay coefficient of variation < 3%), and accuracy (average recovery > 98%). Changes in serum cortisol, glucose and chloride were measured in rainbow trout and lake trout following an acute 1-min handling stressor. In both species, cortisol levels rose rapidly to peaks of approximately 300 ng/ml by 1 h post-stress, and slowly returned to baseline over the next 48 h. Glucose levels rose faster in rainbow trout than in lake trout but reached similar peak levels of approximately 135 mg/dl at 6 h post-stress in both species. Chloride levels declined between 1 and 3 h in both species, although rainbow trout showed a greater and more prolonged hypochloremia than lake trout. Within 1 week following administration of the stressors, 19 rainbow trout ( ∼ 17% of the fish in the experiment) and 2 lake trout ( ∼ 2%) died. Most of the fish which died had been sampled when their serum chloride levels were low, suggesting that the survival rates of stocked trout may be increased if they are rehandled and stocked before or after this period of hypochloremia.