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

Clin Diagn Lab Immunol. 2003 Jan;10(1):133-9. doi: 10.1128/cdli.10.1.133-139.2003.

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.

Publication types

  • Validation Study

MeSH terms

  • Arthritis, Juvenile / immunology
  • Arthritis, Rheumatoid / immunology
  • Blood Cells
  • Case-Control Studies
  • Cytokines / analysis*
  • Humans
  • Immunoassay / methods
  • Lymphocyte Activation / drug effects
  • Lymphocyte Activation / immunology
  • Lymphocytes / chemistry*
  • Lymphocytes / drug effects
  • Lymphocytes / immunology
  • Phytohemagglutinins / pharmacology
  • Protein Array Analysis / methods*
  • Reference Standards
  • Reproducibility of Results
  • Tetanus Toxoid / pharmacology

Substances

  • Cytokines
  • Phytohemagglutinins
  • Tetanus Toxoid