Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells

Abstract

Cytokines secreted by cells of the immune system can alter the behavior and properties of immune or other cells. At a site of inflammation, sets of cytokines interact with immune cells, and their combined effect is often more important than the function of one isolated component. Conventional techniques, such as enzyme-linked immunosorbent assays, generally require large quantities of cells to characterize a complete cytokine profile of activated lymphocytes. The Bio-Plex system from Bio-Rad Laboratories combines the principle of a sandwich immunoassay with the Luminex fluorescent-bead-based technology. We developed a multiplex cytokine assay to detect different cytokines simultaneously in culture supernatant of human peripheral blood mononuclear cells stimulated with antigen and with mitogen. Fifteen human cytokines (interleukin 1alpha [IL-1alpha], IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-18, gamma interferon, and tumor necrosis factor alpha) were validated with a panel of healthy individuals, rheumatoid arthritis patients, and juvenile idiopathic arthritis patients. Comparing the multiplex assay with a regular enzyme-linked immunosorbent assay technique with this donor panel resulted in correlation coefficients for all cytokines ranging from 0.75 to 0.99. Intra-assay variance proved to be less then 10%, whereas interassay variability ranged between 10 and 22%. This multiplex system proved to be a powerful tool in the quantitation of cytokines. It will provide a more complete picture in differences between activated lymphocyte cytokine profiles from healthy individuals and those from patients with chronic inflammatory diseases.

FIG. 1.

Standard curves for recombinant cytokines. Data were generated by combining a threefold dilution of the standards starting at 22,500 pg/ml, coupled beads, and biotinylated antibodies in a 70-μl matrix solution incubated for 20 min. SA-PE was added after two washes and incubated for 10 min. After an additional wash, the median fluorescence intensity was measured with the Bio-Plex system. Standard curves were calculated with Bio-Plex Manager software by a five-parameter regression formula.

FIG. 2.

Specificities of cytokine determination in a multiplex system. A complete set of 15 microspheres were mixed with all biotinylated antibodies and one single standard (2,500 pg/ml). (A) MFI was measured using the Bio-Plex system for IL-10 at 2,500 pg/ml; (B and C) comparison of Bio-Plex and ELISA results. Shown are regression lines for IL-1β (correlation coefficient, 0.96) (B) and IL-6 (correlation coefficient, 0.85) (C).

FIG. 3.

Cytokine profiles of in vitro-stimulated human lymphocytes. PBMCs from healthy controls (HC), (RA), and JIA patients were stimulated for 96 h with medium only, TT, or PHA. Supernatants were collected and cytokine levels were measured by the Bio-Plex system. The mean cytokine concentration (picograms per milliliter) of the TT stimulation is plotted at two-thirds of the logarithmic scale. Yellow squares indicate that the corresponding cytokine is undetectable (lower detection limit of the assay); black squares indicate the upper detection limit of the cytokine (see Table 2); blank squares indicate that the cytokine was not tested.