Evaluation of Enzyme Immunoassay and Radioimmunoassay Methods for the Measurement of Plasma Oxytocin

Abstract

Objective There is increased interest in measuring peripheral oxytocin levels to better understand the role of this peptide in mammalian behavior, physiology, and disease. The purpose of this study was to compare methods for plasma oxytocin measurement using a commercially available enzyme immunoassay (EIA) and radioimmunoassay (RIA), to evaluate the need for sample extraction, and to assess the immunospecificity of the assays. Methods Oxytocin was measured in extracted and unextracted human plasma samples (n = 39). Oxytocin and its degradation products were separated by high performance liquid chromatography and gel filtration chromatography and then assayed by EIA or RIA to identify oxytocin immunoreactive peaks. Results Without extraction, plasma measured by EIA was more than 100-fold higher than in extracted plasma, and the correlation between oxytocin levels in extracted and unextracted plasma was minimal (Spearman’s rho = −0.10, p = 0.54). Using the RIA, most samples (> 90%) were below the level of detection with or without extraction. Following chromatographic fractionation of sample extracts, multiple immunoreactive products were found to be present in addition to oxytocin, which casts doubts on the specificity of the assays. Conclusions Changes in oxytocin levels have been reported in social and behavioral challenge studies. This study indicates that sample extraction is necessary to obtain valid assay results. Changes in oxytocin degradation products are likely to contribute to the previously observed responses in circulating oxytocin levels to behavioral and social challenge. There is a critical need for validated and reliable methods to measure oxytocin in biological samples.

Plasma Oxytocin and Arginine-Vasopressin Levels in Children with Autism Spectrum Disorder in China: Associations with Symptoms

“…The methods of OXT measurement used in these studies were radioimmunoassay (RIA) and enzyme-linked immunoassay (ELISA) without sample extraction. In the present study, ELISA was conducted to measure OXT levels following plasma sample extraction, which has been suggested to be more sensitive and accurate than RIA or ELISA without extraction [31,32]. Therefore, the differences in assay methods for OXT may help to explain the inconsistent findings.…”

“…In order to make accurate measurements, all samples were extracted with acetone and petroleum ether according to the Product Manual and published methods [31,32]. Extraction is necessary to eliminate interfering substances in the samples and to increase the low plasma concentrations of OXT and AVP for accurate measurements within the range of the assay.…”

Natural variation in maternal sensitivity is reflected in maternal brain responses to infant stimuli.

“…Analyses include sample extraction (filtration of OT from its degradation products), which, according to Szeto et al (2011), is necessary to obtain valid assay results through getting rid of multiple immunoreactive products that cast doubt on the specificity of the assays.…”

Mood and Oxytocin Blood Levels in Physically Active Women with and without Oral Contraceptive Use in Relation to Seasonal Daylight Variation

“…A repeated assessment in this specific location with marked seasonal variability in daylight hours was performed, and the result showed that seasonal variation of Oxytocin probably was not influenced by the ent the levels also as ratios of the mean values, which show that physically active women with oral contraceptives have more than 1.1 to 1.5 times higher levels compared to women without oral contraceptives ( Table 2). Oxytocin is known to be released into peripheral circulation in a pulsative fashion, as are most of the body's hormones, where it is assumed to have a relatively brief half-life [48] estimated between 1-2 minutes [49] and 3-9 minutes [50] hence it was important to collect the blood sample at the same time; in this study, all blood samples were taken between 07:30 h and 08:30 h. To provide as comparable and as calm a reception as possible the same routines and technical assistance were used.…”