Measuring multiple hormones from a single water sample using enzyme immunoassays

Many aquatic species, such as teleosts, release into the water and detect multiple bioactive substances to assist in schooling, migration, alarm reactions, and to stimulate behavioral and physiological responses during reproduction and in parent–offspring interactions. Understanding the complex relationship between hormones, behavior and their function in communication requires the simultaneous examination of multiple circulating hormones. However, repeated blood sampling within a short time period is not possible in smaller animals without impacting the very behaviors under investigation. The non-invasive technique of collecting and measuring hormone values in holding water using either radioimmunoassay (RIA) or enzyme immunoassay (EIA) is becoming widely used in teleost research. Commercial assay kits in particular enable rapid and reliable data generation, yet their assay buffers are often specific and potentially incompatible with each other, which can hinder measuring multiple hormones from the same sample. We present here the validation and application of a “nested” elution technique we developed that allows for repeated sampling of multiple reproductive hormones – testosterone (T), 17β-estradiol (E2), progesterone (P), prostaglandin F2α (PGF) and 11-ketotestosterone (11KT) – from individual samples of animal holding water by using commercial EIA systems. Our results show that when using appropriate controls to account for possible technical and biological confounds, this technique provides a powerful new tool for research in aquatic endocrinology and physiology.