Validation,use, and disadvantages of enzyme-linked immunosorbent assay kits for detection of cortisol in channel catfish,largemouth bass,red pacu,and golden shiners

Measurement of cortisol response is an important tool to asses stress in fisheries research. Radioimmunoassay (RIA) is a common method for the measure of cortisol in fish. Use of enzyme-linked immunosorbent assays (ELISA) to detect cortisol would eliminate health hazards, costs of handling radioisotopes, and the short stability time associated with RIA. Enzyme-linked immunosorbent assays have been used for the determination of cortisol in several fish species. However, the ELISA method of cortisol determination in fish lacks proper validation testing. We conducted validation procedures for multiple commercial cortisol ELISA kits and compared the results to RIA. The assays were tested for four species: (1) channel catfish Ictalurus punctatus, (2) largemouth bass Micropterus salmoides, (3) red pacu Piaractus brachypomus, and (4) golden shiners Notemigonus crysoleucas. We evaluated the ELISA methods against RIA, and determined that at least one kit is suitable (accuracy: mean recovery of spiked samples, 102.8%; reproducibility: interassay coefficient of variation < 10.5% for all species; precision: intra-assay coefficient of variation < 16.8% for all species; linearity: R ² > 0.96 for all species) for the measurement of cortisol response in fish and comparative determination of stress. All of the ELISA assay results varied by more than 10% from the cortisol concentrations detected by the RIA. The high variability of the kit results indicates that commercial ELISA kits could be utilized for qualitative determination of cortisol in fish, but should be fully validated in each laboratory for each species before being used for research.