Validation of a Time‐Resolved Fluoroimmunoassay for Measuring Plasma Cortisol in Channel Catfish Ictalurus punctatus

Abstract.— A time‐resolved fluoroimmunoassay (TR‐FIA) marketed for measuring cortisol in human sera was evaluated and validated for use in the quantification of plasma cortisol concentrations of channel catfish Ictalurus punctatus. Time‐resolved fluoroim‐munoassays provide non‐isotopic alternatives to the use of radioimmunoassays (RIA). The evaluated TR‐FIA satisfied strict criteria of precision (intra‐assay coefficients of variation (CV) < 7%) and reproducibility (inter‐assay CV ≤ 9%). Accuracy of the TR‐FIA, calculated as the percent of exogenous cortisol recovered from spiked catfish plasma, averaged 99.5%. Assay sensitivity (minimum detection limit) in catfish plasma was 1.2 ng/mL, and the displacement curve for serially diluted channel catfish plasma paralleled the cortisol standard curve. Plasma cortisol concentrations of channel catfish in the presence and absence of a confinement stressor were used to characterize the im‐munoreactive cortisol measurable by TR‐FIA, and compared favorably to RIA values for the same samples (r2= 0.95, P < 0.001).