Improved Sensitivity of Colorimetric Compared to Chemiluminescence ELISAs for Cytokine Assays

Abstract

Cytokines are often measured using ELISAs and chemiluminescence (CMIL) is reported to exhibit increased sensitivity compared to colorimetric (COL) assays. CMIL also has a wider dynamic detection range. We sought to directly compare ELISAs for measuring human TNF and IL-8 using CMIL or COL. CMIL substrates with glow fluorescence were obtained from 4 different commercial sources while the COL substrate was TMB. ELISAs for TNF and IL-8 were run under identical conditions and the standard curve extended from 0.5 to 4000 pg/ML. The COL substrate demonstrated a sigmoid shaped curve when plotted on a log-linear scale while the CMIL continued to increase up to the highest concentration. Both substrates were modeled most accurately by a 4 parameter equation with R values >0.99. The standard curves for both the IL-8 and TNF demonstrated a lower limit of detection (LLD) for the COL comparable to the CMIL detection system. To precisely define the LLD quadruplicate blanks were run and the mean plus 4 standard deviations were used. By these criteria, the COL assay routinely had a LLD of <1.5 pg/ML which was better than any of the CMIL substrates. Our data demonstrate the COL assays have the same or better sensitivity than CMIL and are significantly less expensive.

Keywords:

• Cytokines
• ELISA
• Immunoabsorbent assay
• Chemiluminescence
• TMB
• Chemokines

Acknowledgments

The work was supported, in part, by NIH grants GM 44918, GM 50401, and GM 62119.