Single molecule measurements of tumor necrosis factor α and interleukin-6 in the plasma of patients with Crohn's disease

The quantitative measurement of inflammatory cytokines in blood has been limited by insufficient sensitivity of conventional immunoassays. This limitation has prevented the widespread clinical monitoring of cytokine concentrations in chronic inflammatory diseases. We applied a sensitive, single molecule detection technology to measure TNF-alpha and IL-6 in the plasma of patients with Crohn's disease (CD), before and after treatment with anti-TNF-alpha therapy. Plasma from 17 patients with CD was collected prior to initiation of anti-TNF-alpha therapy, and the Crohn's disease activity index (CDAI) was determined for each patient. A sub-set of these patients returned for follow up 12 weeks after treatment started. Plasma from age- and gender-matched controls was also collected. Digital ELISAs were developed for TNF-alpha and IL-6, and the plasma concentrations of these cytokines were determined using digital ELISA. The limits of detection of the TNF-alpha and IL-6 digital ELISAs were 0.008 pg/mL and 0.006 pg/mL, respectively. Both cytokines were detected in all samples using digital ELISA and the concentrations of TNF-alpha. and IL-6 in the plasma of patients with CD were (3.6 +/- 0.9) pg/mL and (10.9 +/- 112) pg/mL, respectively. TNF-alpha levels in patients and healthy controls were not significantly different, but the IL-6 levels in plasma were significantly elevated in patients compared to controls. After therapy, the mean reduction of the concentrations of free TNF-alpha and IL-6 were 46% and 58%, respectively. Digital ELISA provided the first quantitative measurements of TNF-alpha and IL-6 concentrations in the plasma of all patients in a population with CD. The changes in cytokine concentrations after therapy-which could be quantified because of the high sensitivity of digital ELISA-could be used for monitoring therapeutic efficacy. (C) 2011 Elsevier B.V. All rights reserved.