Comparison of serum cytokine ELISA measurements between laboratory sites
Authors: Wolfe Anthony S. , Brown Shyretha D. , Ungaro Corey T. 
Published: April 2020

Serum cytokine values are used as indicators to examine a variety of health and performance‐related interventions in physiology. However, commonly utilized enzyme‐linked immunosorbent assay (ELISA) test kits are often not comparable against one another, making interpretation of absolute values between studies and throughout the literature difficult. To address this concern, the purpose of this study was to determine if serum cytokine (IL‐1β, IL‐6) ELISA measurements are comparable across two laboratory sites (Site 1: Frisco, TX and Site 2: Barrington, IL) using the same samples and methods. At Site 1, thirty‐three blood samples were collected from the antecubital vein of fasted professional American football players, and 300 μL were aliquoted into 6 cryovials after clotting and centrifugation and frozen immediately at −80°C. Half of the serum aliquots for the 33 samples were shipped from Site 1 and received frozen to Site 2. ELISA vs ELISA between sites testing procedure was standardized for spectrophotometer (Biotek), ELISA manufacturer (Sigma‐Aldrich), sample handling, lot number, and time of analysis. All assay procedures were performed per the manufacturer instructions with no technical issues. Both sites used four parameter non‐linear logistical standard curves with a r2=1 for all assays. Cytokine values are reported as mean ± SD. Site 1 versus Site 2 serum cytokine concentrations were compared using paired samples t‐tests with 95% CI. Coefficient of variation (CV%) was used to make within and between site comparisons. Results indicated that IL‐1β: [n= 25; (Site 2) 38.2 ± 41.7 pg/mL, (Site 1) 33.9 ± 35.2 pg/mL] had an inter‐site CV% of 15.4 and intra‐assay Site 1 and Site 2 CV% of 7.5 and 10.9. IL‐6: [n=33 (Site 2) 341.9 ± 453.3 pg/mL, (Site 1) 417.4 ± 698.1 pg/mL] had an inter‐site CV% of 36.9 and intra‐assay Site 1 and Site 2 CV% of 8.4 and 12.3. Nine IL‐1β samples were lost due to overflow in optical density during analysis. Paired samples t‐tests showed a significant mean difference for IL‐1β [4.2 (95% CI, −8.2 to −0.3) pg/mL, p = 0.034] between sites and no significant mean difference for IL‐6 [75.5 (95% CI, −14.3 to 165.3)] pg/mL, p = 0.096 between sites. Inter‐site CV’s were ~2× the intra‐assay CVs, indicating that caution should be taken when interpreting the absolute values of cytokines measured across laboratories using ELISA assays and from multiple publications. The source of the large inter‐assay variation for IL‐6 was predominately located towards the lower limits of detection, identifying a need for manufacturers to develop higher sensitivity assays across these markers. Future research is warranted to understand possible causes of variability in absolute cytokine values across two laboratories using the same samples and the same ELISA kits.Support or Funding InformationThis study was funded by and the authors are employed by the Gatorade Sports Science Institute, a division of PepsiCo, Inc. The views expressed in this abstract are those of the authors and do not necessarily reflect the position or policy of PepsiCo, Inc.