Metcalfe, Suzanne S., Kroon, Frederieke J., Beale, David J., and Miller, Gabrielle (2018) Development of a validation protocol of enzyme immunoassay kits used for the analysis of steroid hormones in fish plasma. Journal of Experimental Marine Biology and Ecology, 499. pp. 26-34.

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Abstract

The analysis of steroid hormones 17β-estradiol, testosterone and 11-ketotestosterone in fish plasma provide insight into fish reproductive behavior and development. Typically, steroid hormones are analyzed using gold standard approaches, such as radioimmunoassay and liquid chromatography coupled with tandem mass spectrometry. However, these methods are not always available to small-to-medium research facilities due to their large capital outlay, expensive running costs and ongoing maintenance needs, in addition to requiring a costly radioisotope license and skilled staff to handle and perform the assays. One approach being increasingly used by researchers is commercial enzyme immunoassay (EIA) kits (also known as ELISA kits), analyzed with the use of a UV–vis spectrometer. However, with their increased use, many studies fail to validate their EIA kits or fail to report on the kits sensitivity, accuracy and precision. As such, the aim of this paper is to propose a standardized protocol for reporting EIA kit validation. The proposed protocol was then applied to three fish species in order to demonstrate parallelism, accuracy and precision. Specifically, we tested plasma concentrations of 17β-estradiol for the red and black anemonefish Amphiprion melanopus, and 17β-estradiol, testosterone and 11-keto-testosterone for the barramundi Lates calcarifer and the common coral trout Plectropomus leopardus. Steroid hormones were extracted from fish plasma using a liquid-liquid solvent extraction method with ethyl acetate and n-hexane (50:50 v:v). Following extraction, the plasma dilution curves showed good linearity and parallelism, steroid recoveries were found to be within 90–120% accuracy, and precision variance was < 10% in the majority of assays, for the species and steroids measured. Utilization of the proposed standardized validation protocol of EIA kits will assist scientific, fisheries and aquaculture researchers in obtaining reliable steroid hormone measurements, particularly in the field and in smaller research laboratories.

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