Comparative Analysis of Serum Cytokine ELISA and Multiplex Techniques

Abstract

Enzyme-linked immunosorbent assay (ELISA) has been described as the gold standard for serum cytokine analysis. However, multiplex array technologies are increasing in popularity, likely because of their ability to analyze large numbers of analytes with low sample volume compared to ELISA. Few studies have directly compared serum cytokine results from ELISA and Multiplex analytical techniques. Therefore, the purpose of this observational research was to determine if differences exist in serum cytokine concentrations between ELISA and Multiplex techniques. Blood samples were collected from the antecubital vein of professional American football players during a competitive season to monitor biomarkers fluctuations throughout the season. After clotting, blood was centrifuged and three 300 μL aliquots of serum were frozen at -80°C. IL-1β, IL-6, and TNF-α were measured from the exact same serum samples using ELISA (EMD Millipore, Sigma Aldrich) and Multiplex (EMD Millipore, Magpix) kits.

Coefficients of variation (CV%), paired samples t-test with 95% CI, and Pearson’s product-moment correlations were used to compare cytokine analysis techniques. Descriptive statistics are shown as mean ± SD. Results indicated that serum cytokine concentrations were not comparable between ELISA and Multiplex analytical techniques. IL-1β (n=15) displayed significant variability between techniques with a CV% of 119.9 with ELISA reading 36.8 ± 32.4 pg/mL, intra-assay CV%= 7.6 and Multiplex showing 1.1 ± 1.2 pg/mL, intra-assay CV%= 6.8. IL-6 (n=33) demonstrated substantial variability between techniques with a CV% of 126.9 with ELISA reading 340.2 ± 460.5 pg/mL, intraassay CV%= 15.3 and Multiplex showing 11.8 ± 20.2 pg/mL, intra-assay CV%= 4.8. For TNF-α (n=36) all ELISA samples were below the detection limit of 0.31 with the Multiplex detecting 8.8±3.2 pg/mL, intra-assay CV%= 7.9. Moreover, paired samples t-tests showed considerable mean differences between analysis techniques for IL-1β (-35.8 pg/mL, (95% CI, -51.6 to -19.9) t(17) = -4.8, p < 0.001) and IL-6 (-328.4 pg/mL (95% CI,-490.0 to -166.7), t(32)= -4.1, p < 0.001.

Pearson’s correlation between ELISA and Multiplex was not significant for IL-1β [r(13) = 0.44, p = 0.068] or IL-6 [r(31) = 0.25, p = 0.157]. The results suggest that ELISA and Multiplex outcomes were not comparable and the differences in blocking agents used may be the source of erraticism. According to the results from this study, comparisons between analytical techniques should be avoided and more work is needed to determine the cause of these large discrepancies seen between methods.