Validation of a Commercially Available Enzyme ImmunoAssay for the Determination of Oxytocin in Plasma Samples from Seven Domestic Animal Species

Abstract

The neurohormone oxytocin (OT) has a broad range of behavioral effects in mammals. It modulates a multitude of social behaviors, e.g., affiliative and sexual interactions. Consequently, the OT role in various animal species is increasingly explored. However, several issues have been raised regarding the peripheral OT measurement. Indeed, various methods have been described, leading to assay discrepancies and inconsistent results. This highlights the need for a recognized and reliable method to measure peripheral OT. Our aim was to validate a method combining a pre-extraction step, previously demonstrated as essential by several authors, and a commercially available enzyme immunoassay (EIA) for OT measurement, using plasma from seven domestic species (cat, dog, horse, cow, pig, sheep, and goat). The Oxytocin EIA kit (EnzoLifeSciences) was used to assay the solid-phase extracted samples following the manufacturer's instructions with slight modifications. For all species except dogs and cats, concentration factors were applied to work above the kit's sensitivity (15 pg/ml). To validate the method, the following performance characteristics were evaluated using Validation Samples (VS) at various concentrations in each species: extraction efficiency via spiking tests and intra- and inter-assay precision, allowing for the calculation of total errors. Parallelism studies to assess matrix effects could not be performed because of too low basal concentrations. Quantification ranges and associated precision profiles were established to account for the various OT plasma concentrations in each species. According to guidelines for bioanalytical validation of immunoassays, the measurements were sufficiently precise and accurate in each species to achieve a total error ≤30% in each VS sample. In each species, the inter-assay precision after 3 runs was acceptable, except in low concentration samples. The linearity under dilution of dogs and cats' samples was verified. Although matrix effects assessments are lacking, our results indicate that OT plasma levels can reliably be measured in several domestic animal species by the method described here. Studies involving samples with low OT plasma concentrations should pay attention to reproducibility issues. This work opens new perspectives to reliably study peripheral OT in a substantial number of domestic animal species in various behavioral contexts.

Working Smarter Not Harder: Oxytocin Increases Domestic Dogs’ (Canis familiaris) Accuracy, but Not Attempts, on an Object Choice Task

“…Such wide variability in OT levels in plasma is not uncommon after intranasal administration and has also been reported in monkeys for instance (Lee et al, 2018). Indeed, a variability of approximately 50% of mean basal plasma OT concentrations has been previously reported within populations from several species, regardless of exogenous OT administration (Bienboire-Frosini et al, 2017). Crockford et al (2014) precisely described and explicated the general variability of the oxytocinergic system within species.…”

“…Furthermore, it has recently been suggested that the neuro-behavioral effects of intranasally administered OT may stem from a peripheral mechanism of action, following results from a study by Lee et al (2018) which demonstrated highly variable timings and extents that both intranasally and intravenously administered OT reached the CSF of monkeys. Temesi et al (2017) and Romero et al (2014) also demonstrated an increase in OT measured in dog blood following intranasal application, however, as these studies used varying methods, doses, and small sample sizes, the current study will extend upon these findings in a larger sample of dogs using recently validated methods (Bienboire-Frosini et al, 2017; MacLean et al, 2017), as stated hereunder in the “Materials and Methods” section.…”

“…The global procedure of extraction/concentration and assay was first internally validated on seven Quality Control samples of dog plasma from an external study to assess the precision and the accuracy of the whole method through the kit’s dynamic range: the mean precision was 12.0% and the mean recovery was 98.3%. Of note, some of the authors have recently published a validation of another Oxytocin ELISA kit from Enzo Life Sciences (Bienboire-Frosini et al, 2017) to assay OT in dog’s plasma. This kit was initially used in this study but unfortunately lead to too many results under the limit of detection.…”

The Impact of Equine-Assisted Therapy on Equine Behavioral and Physiological Responses

“…The conclusions of that study relied on HRV and the plasmatic concentrations of cortisol and oxytocin before, during, and after EAT sessions. The assessment of oxytocin concentrations in equine plasma through enzyme immunoassay requires an extraction phase, as described by Bienboire-Frosini et al [38]. This procedure was not performed in the previously cited study [20].…”

TDAG51 is a crucial regulator of maternal care and depressive-like behavior after parturition

“…The ΔΔCT value was calculated by the following equation: (ΔCT (each sample) —ΔCT (nonpregnant wild-type female) ). An ELISA was performed as previously described [73]. Briefly, sera were collected from the mice, centrifuged, and subjected to ELISA.…”