Specificity of plasma oxytocin immunoassays: A comparison of commercial assays and sample preparation techniques using oxytocin knockout and wildtype mice

• Published in: Psychoneuroendocrinology Vol. 132; p. 105368
• Main Authors: Gnanadesikan, Gitanjali E., Hammock, Elizabeth A.D., Tecot, Stacey R., Carter, C. Sue, MacLean, Evan L.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.10.2021
• Subjects: Animals; Biological Assay; ELISA; Extraction; Immunoassay; Knockout; Mice; Mice, Knockout; Oxytocin; Oxytocin - blood; Plasma; Sample matrix; Specimen Handling; Immunoassay; Sample matrix; Extraction; Oxytocin; Knockout; ELISA
• ISSN: 0306-4530; 1873-3360
• DOI: 10.1016/j.psyneuen.2021.105368

Oxytocin has garnered much interest due to its role in affective states, social behaviors, and diverse physiological functions. However, approaches for measuring endogenous oxytocin concentrations have generated considerable controversy and debate. Common procedures for measuring oxytocin often produce uncorrelated results, and the detected concentrations frequently vary across two orders of magnitude. These findings have led some researchers to argue that immunoassays of plasma oxytocin may be unreliable and nonspecific, particularly when samples are not first processed using an extraction procedure. Here, we assess the specificity of oxytocin immunoassays using plasma samples from wildtype (WT) and oxytocin knockout (KO) mice. Plasma samples from both genotypes were measured using immunoassay and were measured with or without a solid-phase extraction. Using a commercially available kit from Arbor Assays, we demonstrate that both techniques generate a clear contrast between genotypes, with wildtype samples containing high concentrations of oxytocin (unextracted mean = 468 pg/ml; extracted mean = 381 pg/ml), while knockout samples measured below the lower limit of detection. Analytical validations demonstrated good parallelism and spike recovery for both methods. Furthermore, the same wildtype samples measured with both procedures were highly correlated (r = 0.95), although unextracted samples measured at significantly higher concentrations (p = 2.0 ×10−7, Cohen’s d = 2.65). To test the generalizability of these results across immunoassay kits, we performed additional assays with kits from Cayman Chemical and Enzo Life Sciences. The Cayman Chemical kit produced results similar to Arbor Assays with a clean signal differentiating WT and KO plasma, both with and without an extraction step. The Enzo kit also differentiated the genotypes, with correlation between extracted and unextracted samples, but was considerably more susceptible to interference without the extraction, as evidenced by false positive signal in KO plasma samples. The extent to which these results generalize to other species remains unknown and challenging to assess. [Display omitted] •Mouse plasma oxytocin can be reliably measured without extraction by immunoassay.•Unextracted plasma samples must be diluted for accurate measurement by immunoassay.•With the reported methods, extracted and unextracted samples are highly correlated.•Results are dependent on the immunoassay, potentially due to antibody differences.•Some commercially available assay kits are susceptible to matrix interference.