Abstract

VELASCO-SANTAMARIA1, Yohana  and  CRUZ-CASALLAS, Pablo. METHODOLOGY FOR DETERMINATION OF PLASMA CORTISOL IN FISH USING COMPETITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA). Rev.MVZ Cordoba [online]. 2007, vol.12, n.1, pp.869-877. ISSN 0122-0268.

Objective. To determine plasma cortisol procedure in fish using competitive enzyme-linked immunosorbent assay (ELISA). Materials and methods. Two plasma samples of juveniles rainbow trout Oncorhynchus mykiss were analized by using ELISA human kit for cortisol assay. For standard curve calibration seven standard solutions of cortisol in human plasma (0, 20, 50, 100, 200, 400 and 800 ng.ml-1) were used. For the recovery test 50, 100 and 200 ng.ml-1 standard solutions of cortisol were used; for the linearity test four dilutions of the fish plasma samples (1/2, 1/4, 1/8 and 1/16) were prepared. To each well fish plasma samples and standard solutions were added and its content were conjugated to peroxidase and subsequently enzyme substrate was added. The enzymatic reaction was stopped by addition of phosphoric acid 0.5 M and the absorbance was read at 450 nm. The accuracy of the pipetting procedure was assessed previously. The recovery and linearity percentages, the standard curve and parallelism were determined. Results. The standard curve showed a high correlation coefficient (r2 = 0.998). The cortisol concentration of two samples fluctuated between 64 and 72 ng.ml-1. Only the 200 ng.ml-1 standard solution showed a recovery percentage superior to 80%; in contrast, in 50 and 100 ng.ml-1 the recovery percentage fluctuated between 52 and 71%. In the dilution of 1/2 to 1/8 were observed a good linearity (86 to 168%); the samples showed parallelism with the standard curve. Conclusions. The use of human plasma cortisol for ELISA procedure is an accuracy and efficiency test for fish cortisol plasma determination.

Keywords : Cortisol; ELISA; fish; Oncorhynchus mykiss; rainbow trout; plasma.

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