Coastal Sciences, Gulf Coast Research Laboratory
Ocean Science and Engineering
Sharks and rays are popular species used in wildlife ecotourism and aquariums to educate the public on the behavior, ecology and conservation challenges of elasmobranchs. To understand long-term physiological health and welfare under varying social and husbandry conditions, we developed and validated an enzyme immunoassay (EIA) to measure stress/ionoregulatory hormones in managed and semi-free range southern rays (Hypanus americanus). Banked serum and interrenal samples from 27 female rays managed at Disney’s The Seas with Nemo and Friends® and Castaway Cay were used to evaluate measurement of 1α-hydroxycorticosterone (1αOHB) relative to corticosterone (B). Although commercial EIAs are available for B, those tested exhibit only low relative cross-reactivity to 1αOHB (3-5%). To improve measurement of 1αOHB, we developed a monoclonal antibody using a synthesized 1αOHB-derivative for evaluation using high-performance liquid chromatography (HPLC) and EIA. Relative displacements of cross-reactant compounds showed that the antibody had good sensitivity for the target antigen 1αOHB, and low sensitivity to related steroids (desoxycorticosterone and B), but greater sensitivity to 11-dehydrocorticosterone. Tests of competitive vs. noncompetitive EIA formats, reagent titration, and incubation times of the antibody and conjugate were used to optimize sensitivity, repeatability and precision of measured 1αOHB in standards and samples (4 ng/ml, 90% binding). Tests of sample pre-treatment (pH adjustment) and extraction with varying solvent polarity were used to optimize measurement of 1αOHB in
General and Comparative Endocrinology
Wheaton, C. J.,
Mylniczenko, N. D.,
Rimoldi, J. M.,
Gadepalli, R. S.,
O'Hara, B. R.,
Evans, A. N.
(2018). Challenges, Pitfalls and Surprises: Development and Validation of a Monoclonal Antibody for Enzyme Immunoassay of the Steroid 1α-Hydroxycorticosterone In Elasmobranch Species. General and Comparative Endocrinology, 265, 83-89.
Available at: https://aquila.usm.edu/fac_pubs/16621