5 years later: irisin detection still an issue

Abstract

Since the discovery of irisin in early 2012 (1), considerable attention has been directed toward understanding the physiological role of this circulating myokine in humans. Animal studies demonstrating important metabolic benefits have been convincing (1, 2), but whether these findings translate to humans remains unclear, and many discrepant results have been reported (3, 4, 5). A tremendous limiting factor continues to be the quality and accuracy of existing commercial enzyme-linked immunosorbent assays (ELISAs) for quantifying circulating irisin levels. With growing interest in this field, resulting in over 500 publications since 2012, we feel it is important to reinforce some of the limitations of the currently available assays and the necessary steps forward, as well as share our own concerning results regarding the Phoenix Pharmaceuticals and Adipogen ELISAs, two of the most widely used assays to date (3). Our group intended to quantify circulating irisin levels in young healthy adults in response to exercise; however, our own validation testing with both ELISAs has been less than optimal. Using the 1st-generation Phoenix Pharmaceuticals EK-067-29 assay, we observed very high coefficients of variation (CVs) of 13.2% and 13.8% for 2 separate human plasma samples (17 duplicates each, loaded interchangeably across the plate). Phoenix Pharmaceuticals subsequently improved the specificity of the biotinylated peptide in the 2nd generation of this assay (current version), yielding concentrations much closer to the ‘true’ circulating irisin levels, determined using tandem mass spectrometry (6). While this seemed a positive development, we remain dissatisfied with the performance of this assay, reporting CVs of 15.2% and 11.0% for 2 samples (13 duplicates each). We further tested the same 2 samples with the Adipogen ELISA (AG-45A-0046YEK-Kbib1) (10 duplicates each) and observed even higher CVs of 21.4% and 11.1% (Table 1). It should be emphasized that the irisin concentrations obtained from the Adipogen assay are considerably higher than the expected range (6): irisin values measured with the Adipogen assay were 477-fold higher than those of the Phoenix assay in the same individual (Table 1).