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METODOLOGÍA PARA LA DETERMINACIÓN DE CORTISOL PLASMÁTICO EN PECES USANDO LA PRUEBA DE INMUNOENSAYO ENZIMÁTICO (ELISA)

REVISTA MVZ CORDOBA 01/2007;
Source: DOAJ

ABSTRACT Objective. To determine plasma cortisol procedure in fish using competitive enzymelinkedimmunosorbent assay (ELISA). Materials and methods. Two plasma samplesof juveniles rainbow trout Oncorhynchus mykiss were analized by using ELISA humankit for cortisol assay. For standard curve calibration seven standard solutions ofcortisol in human plasma (0, 20, 50, 100, 200, 400 and 800 ng.ml-1) were used. Forthe 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 addedand its content were conjugated to peroxidase and subsequently enzyme substratewas added. The enzymatic reaction was stopped by addition of phosphoric acid 0.5 Mand the absorbance was read at 450 nm. The accuracy of the pipetting procedurewas assessed previously. The recovery and linearity percentages, the standard curveand parallelism were determined. Results. The standard curve showed a highcorrelation coefficient (r2 = 0.998). The cortisol concentration of two samplesfluctuated between 64 and 72 ng.ml-1. Only the 200 ng.ml-1 standard solution showeda recovery percentage superior to 80%; in contrast, in 50 and 100 ng.ml-1 therecovery percentage fluctuated between 52 and 71%. In the dilution of 1/2 to 1/8were observed a good linearity (86 to 168%); the samples showed parallelism withthe standard curve. Conclusions. The use of human plasma cortisol for ELISAprocedure is an accuracy and efficiency test for fish cortisol plasma determination

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