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 fluoroimmunoassays provide non-isotopic alternatives to the use of radioimmunoassays (RIA). The evaluated TR-FIA satisfied strict criteria of precision (intra-essay 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 immunoreactive cortisol measurable by TR-FIA, and compared favorably to RIA values for the same samples (r2 = 0.95, P < 0.001).