A fully-automated, six-plex single molecule immunoassay for measuring cytokines in blood

Abstract

We report a system and assay for performing fully-automated measurement of 6 proteins simultaneously with single molecule sensitivity. The system combines handling of samples, reagents, and consumables, with a module for imaging single molecule arrays (Simoa) to enable immunoassays that have high sensitivity (~fg/mL), are multiplexed, and are fully-automated. A 6-plex cytokine Simoa assay for IL-6, TNF-α, GM-CSF, IL-10, IL-1β, and IL-1α was developed on the system. The assays had limits of detection in the range 0.01-0.03pg/mL, and the average imprecision (CV) of the Simoa signal was 4.2%. This assay was used to measure the concentrations of these cytokines in the plasma of patients with Crohn's Disease (CD), before and after treatment with anti-TNF-α antibody drugs, and in the serum of Type 1 diabetics. Concentrations of TNF-α and IL-6 in the CD samples determined using the fully-automated, multiplex Simoa assay had good correlation with the manual, single-plex assays previously reported. Drug treatment caused reductions in the mean concentration of all 6 cytokines in the plasma of CD patients. The concentrations of 4 cytokines were significantly higher in diabetics compared to healthy controls. The system could enable the widespread, multiplexed measurement of protein biomarkers with low abundance.

Keywords: Automation; Digital ELISA; Multiplexed immunoassays; Simoa.

Figure 1

Schematic illustrating the work flow of “sample-in to result-out” on the automated, multiplexSimoa system.

Figure 2

Plots of average number of enzymes per bead (AEB) against concentration (0–1 pg/mL) of 6 cytokines spiked into bovine serum from the first run of three measured using the 6-plex assay performed on the automated Simoa system. The inset plots show the curves across the entire range (0–30 pg/mL). The solid lines are fits to the data using a 4 parameter logistic equation; all fits had R² values > 0.99. The 10 pg/mL data point for GM-CSF was omitted from that fit to improve the fit quality of the low concentration data. As for all plots, error bars are shown based on one standard deviation of triplicate measurements. If the error is smaller than the symbol plotted, error bars are not shown.

Figure 3

Scatter plots of the concentrations determined using Simoa of 6 cytokines in the plasma of CD patients and matched healthy controls. The thick and thin solid lines are mean concentrations and standard error means (SEM) for that population. The dotted lines represent the average LOD of each cytokine in the 6-plex Simoa assay.

Figure 4

Plots showing correlation between concentration of IL-6 and TNF-α in the plasma of CD patients determined using the fully-automated, 6-plex Simoa assay (Table S6) and the manual, single-plex Simoa assay previously reported.

Figure 5

Scatter plots of the concentrations determined using Simoa of 6 cytokines in the serum of patients with Type 1 diabetes and healthy controls. The thick and thin solid lines are mean concentrations and SEM for that population. The dotted lines represent the average LOD of each cytokine in the 6-plex Simoa assay.