Challenges, pitfalls and surprises: development and validation of a monoclonal antibody for enzyme immunoassay of the steroid 1 alpha -hydroxycorticosterone in elasmobranch species

Publication Type:
Journal Article
Year of Publication:
Catharine J. Wheaton, Natalie D. Mylniczenko, John M. Rimoldi, Rama S. V. S. Gadepalli, R. Hart, Bobbi R. O'Hara, Andrew N. Evans
General and Comparative Endocrinology
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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[registered trademark] and Castaway Cay were used to evaluate measurement of 1[alpha]-hydroxycorticosterone (1[alpha]OHB) relative to corticosterone (B). Although commercial EIAs are available for B, those tested exhibit only low relative cross-reactivity to 1[alpha]OHB (3-5%). To improve measurement of 1[alpha]OHB, we developed a monoclonal antibody using a synthesized 1[alpha]OHB-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 1[alpha]OHB, 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 1[alpha]OHB 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 1[alpha]OHB in <1 ml (serum) or 1 g (interrenal) samples. HPLC analysis revealed the 1[alpha]OHB EIA to be superior for measurement of 1[alpha]OHB compared to use of a B EIA with or without HPLC fractioning. Results may prove useful for extrapolation to guide best practices for 1[alpha]OHB measurement in other elasmobranch species. Improved measurement of stress/ionoregulatory hormones in sharks and rays will be important for many aspects of collection, transport, medical treatment in aquaria and conservation management of these charismatic and ecologically important species. (C) 2018 Elsevier Inc. All rights reserved.


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