Flow cytometric microsphere-based immunoassay: analysis of secreted cytokines in whole-blood samples from asthmatics

Abstract

The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microsphere-based assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-gamma), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data (r > 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 (P = 0.003) and less IFN-gamma (P = 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-gamma than that of atopic nonasthmatic subjects (P = 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases.

FIG. 1

Dot plots of PC5 fluorescence (ordinate) versus FITC fluorescence (abscissa) for FMBA. Six cytokines were simultaneously measured in unstimulated whole blood (A) and in PMA- and ionomycin-stimulated whole blood (B). Dots in gate Q represent calibration microspheres.

FIG. 2

Standard curves of IL-4, IL-6, IL-10, IL-12, IFN-γ, and TNF-α international standards obtained by FMBA. Assays were run in triplicate; results are means. Curves are presented as mean channel fluorescence versus concentration of standard.

FIG. 3

Follow-up of two IL-6 control samples read on the stored master curve without (open symbols) or with (solid symbols) recalibration by a two-point recalibration procedure.

FIG. 4

Correlation between FMBA and Immunotech ELISA for IL-4, IL-6, IL-10, IL-12, IFN-γ, and TNF-α on 40 samples. Linear regressions were calculated as follows: for IL-4, y = 0.96x − 10.07 and r = 0.982; for IL-6, y = 0.96x − 4.33 and r = 0.987; for IL-10, y = 0.1.16x − 4.15 and r = 0.989; for IL-12, y = 0.92x − 11.43 and r = 0.994; for IFN-γ, y = 1.17x − 8.33 and r = 0.989; for TNF-α, y = 0.93x + 3.38 and r = 0.986.

FIG. 5

Secretion of IL-4, IL-6, IL-10, IL-12, IFN-γ, and TNF-α in PMA- and ionomycin-stimulated whole blood from six nonatopic asthmatic (As), 10 atopic nonasthmatic (At), 30 atopic asthmatic (At As), and 15 nonatopic, nonasthmatic (control) subjects.

FIG. 6

Ratio of IL-4 to IFN-γ production in PMA- and ionomycin-stimulated whole blood from nonatopic asthmatic (As), atopic nonasthmatic (At), atopic asthmatic (At As), and nonatopic, nonasthmatic (control) subjects.

FIG. 7

Percentage of CD3⁺ cells producing IFN-γ in PMA- and ionomycin-stimulated whole blood from nonatopic, nonasthmatic (Control), nonatopic asthmatic (As), atopic nonasthmatic (At), and atopic asthmatic (At As) individuals.