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

-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 TRFIA satisfied strict criteria of precision (intra-assay coefficients of variation (CV) < 7%) and reproducibility (inter-assay CV 5 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-HA, and compared favorably to RIA values for the same samples (6 = 0.95, P < 0.001). Evaluations of stressors and stress responses of cultured fish can lead to improved management practices that minimize stress and thus improve production efficiency. An effective means of assessing the degree of stress in cultured fish is to measure changes in circulating levels of cortisol following administration of a stressor (Mazeaud et al. 1977; Donaldson 1981; Adams 1990; Barton and Iwama 199 1). Accurate assessment of circulating cortisol levels is dependent upon a reliable and repeatable assay. Radioimmunoassays (RIA) are the most common method of quantifying plasma cortisol concentrations. Radioimmunoassays are highly sensitive, accurate and precise; however, the use of radioisotopes diminishes the appeal of this methodology. Alternatively, enzyme-linked immunosorbant assays (ELISA) and other non-isotopic techniques have been used with some success to measure cortisol in fish plasma (Caldwell et al. 1990; Barry et al. 1993). More recently, the use of lanthanum chelates in time-resolved fluoroimmunoassays (TR-FIA) has provided a reliable non-isotopic alternative. The objective of this study was to evaluate and validate a commercially available cortisol TR-FIA for its use in measuring channel catfish Zctulurus punctatus plasma cortisol concentrations. Materials and Methods Twenty fingerling channel catfish of approximately 100 g were acclimated for 7 d in a 535-L aquarium supplied with recirculating water held at 26 C. Following the acclimation period, a basket-confinement stressor was applied. Re-stress blood samples were taken from ten fish and the remaining fish were confined in a plastic basket immersed so that the fish were in contact with each other and could not maintain their orientation. The basket was placed over an airstone to maintain adequate dissolved oxygen. After 2 h, the catfish were anesthetized with MS-222 (tricaine methanesulfonate) at a concentration of 0.2 g/L, and blood was collected from caudal vessels with heparinized syringes. Plasma was separated by centrifugation and stored at 0 Copyright by the World Aquaculture Society 2002