Can antibody-based assays consistently detect differences in feather corticosterone?

Abstract

Measuring corticosterone (Cort) in bird feathers has become increasingly popular as a non-invasive method of obtaining an integrated profile of Cort exposure during the period of feather replacement. Most studies use antibody-based assays to assess Cort levels in feathers [radioimmunoassays (RIA) or enzyme immunoassays (EIA)]. However, it is still unclear whether differences in Cort can be reliably and consistently detected in feathers using antibody-based assays, in part because it is not known how much Cort is present in feathers and antibodies can differ in their ability to detect their antigens. In this study, we tested six commercially available polyclonal Cort antibodies in a feather Cort RIA in nine species. We found that different antisera detected very different levels of Cort in feathers. Additionally, we found that the broad patterns of Cort across species were not the same when measured with different antibodies. Further analysis by mass spectrometry indicated the presence of very little Cort in the feathers of any of the five species tested, suggesting that antibodies were instead binding with Cort metabolites or other substances. These data indicate a potential hidden source of variability when measuring feather Cort with antibody-based tests. The data further suggest caution in cross-species comparisons because patterns seen in feather Cort may reflect artifacts of the specific antibody used in the assay.

Zusammenfassung

Können antikörperbasierte Untersuchungen konsistent Unterschiede im Federkortikosteron nachweisen?

Die Messung von Kortikosteron (Cort) in Vogelfedern erfreut sich als nicht-invasive Methode zunehmender Beliebtheit, um ein Gesamtprofil der Cort-Ausschüttung während des Federwechsels zu erstellen. Die meisten Studien verwenden antikörperbasierte Untersuchungen, um den Cort-Spiegel in Federn zu messen (Radioimmunoassays, RIA, oder Enzymimmunoassays, EIA). Es ist jedoch immer noch unklar, ob Unterschiede im Feder-Cort mit antikörperbasierten Untersuchungen zuverlässig und konsistent nachgewiesen werden können, zum Teil deshalb, weil unbekannt ist, wieviel Cort in Federn enthalten ist und inwiefern Antikörper sich in ihrer Fähigkeit, ihre Antigene zu erkennen, unterscheiden. In dieser Studie untersuchten wir sechs kommerziell erhältliche polyklonale Cort-Antikörper im Feder-Cort-RIA bei neun Vogelarten. Wir fanden heraus, dass die verschiedenen Antiseren sehr unterschiedliche Cort-Mengen in den Federn nachweisen. Darüber hinaus stellten wir fest, dass die allgemeinen Cort-Muster bei den verschiedenen Arten nicht gleich waren, wenn sie mit unterschiedlichen Antikörpern gemessen wurden. Weitere Analysen durch Massenspektrometrie zeigten eine sehr geringe Cort-Menge in den Federn fünf untersuchter Arten, was darauf hindeutet, dass die Antikörper sich stattdessen an Cort-Metaboliten oder anderen Substanzen banden. Diese Daten lassen möglicherweise auf eine versteckte Quelle der Variabilität bei der Messung von Feder-Cort mit antikörperbasierten Tests schließen. Weiterhin rufen die Daten bei artenübergreifenden Vergleichen zur Vorsicht auf, da die in dem Feder-Cort beobachteten Muster Artefakte durch die in den Untersuchungen verwendeten spezifischen Antikörper widerspiegeln können.

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Code availability

Not applicable.

Availability of data and material

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgments

We thank C.R. Lattin for advice on the feather RIA and M. Pokras for providing feathers from the Tufts Veterinary Clinic. We are grateful to Katalin Boroczky and the Cornell Chemical Ecology Core Facility for conducting the LC-MS/MS analyses presented here, and to Meena Harribal for discussions and exploratory analyses. This work was supported by National Science Foundation (NSF) grants IOS-1048529 and IOS-1655269 to LMR, and funds from Cornell University and NSF IOS-1457251 to MNV. All experiments complied with the current laws of the United States.

Funding

This work was supported by NSF grants IOS-1048529 and IOS-1655269 to LMR, and funds from Cornell University and NSF IOS-1457251 to MNV.

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Correspondence to L. Michael Romero.

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Fischer, C.P., Vitousek, M.N. & Romero, L.M. Can antibody-based assays consistently detect differences in feather corticosterone?. J Ornithol (2021). https://doi.org/10.1007/s10336-021-01866-0

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Keywords

  • Feather corticosterone
  • Hypothalamic–pituitary–adrenal axis
  • Glucocorticoids
  • Non-invasive technique