What are oxytocin assays measuring? Epitope mapping, metabolites, and comparisons of wildtype & knockout mouse urine

Oxytocin has become a popular analyte in behavioral endocrinology in recent years, due in part to its roles in social behavior, stress physiology, and cognition. Urine samples have the advantage of being non-invasive and minimally disruptive to collect, allowing for oxytocin measurements even in some wild populations. However, methods for urinary oxytocin immunoassay have not been sufficiently optimized and rigorously assessed for their potential limitations. Using samples from oxytocin knockout (KO) and wildtype (WT) mice, we find evidence of considerable interference in unextracted urine samples, with similar distributions of measured oxytocin in both genotypes. Importantly, although this interference can be reduced by a reversed-phase solid-phase extraction (SPE), this common approach is not sufficient for eliminating false-positive signal on three immunoassay kits. To better understand the source of the observed interference, we conducted epitope mapping of the Arbor Assays antibody and assessed its cross-reactivity with known, biologically active fragments of oxytocin. We found considerable cross-reactivity (0.5–52% by-molarity) for three fragments of oxytocin that share the core epitope, with more cross-reactivity for longer fragments. Given the presence of some cross-reactivity for even the tripeptide MIF-1, it is likely that many small protein metabolites might be sufficiently similar to the epitope that at high concentrations they interfere with immunoassays. We present a new mixed-mode cation-exchange SPE method that minimizes interference—with knockout samples measuring below the assay's limit of detection—while effectively retaining oxytocin from the urine of wildtype mice. This method demonstrates good parallelism and spike recovery across multiple species (mice, dogs, sifakas, humans). Our results suggest that immunoassays of urine samples may be particularly susceptible to interference, even when using common extraction protocols, but that this interference can be successfully managed using a novel mixed-mode cation exchange extraction. These findings imply that previous conclusions based on urinary oxytocin measurements—especially those involving unextracted samples—may need to be reassessed.