Challenges, Pitfalls and Surprises: Development and Validation of a Monoclonal Antibody for Enzyme Immunoassay of the Steroid 1α-Hydroxycorticosterone In Elasmobranch Species

Authors

Catharine J. Wheaton, Disney's Animal Kingdom and The SEAS with Nemo and FriendsFollow
Natalie D. Mylniczenko, Disney's Animal Kingdom and The SEAS with Nemo and Friends
John M. Rimoldi, University of Mississippi
Rama S.V.S. Gadepalli, University of Mississippi
R. Hart, Arbor Assays L.L.C.
Bobbi R. O'Hara, Arbor Assays L.L.C.
Andrew N. Evans, University of Southern MississippiFollow

Document Type

Article

Publication Date

9-1-2018

Department

Coastal Sciences, Gulf Coast Research Laboratory

School

Ocean Science and Engineering

Abstract

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

Comments

© 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0

Publication Title

General and Comparative Endocrinology

Volume

265

First Page

83

Last Page

89

Recommended Citation

Wheaton, C. J., Mylniczenko, N. D., Rimoldi, J. M., Gadepalli, R. S., Hart, R., 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