Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells.
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.
de Jager
Wilco
W
Department of Pediatric Immunology, University Medical Center Utrecht, Wilhelmina Children's Hospital, 3584 EA Utrecht, The Netherlands.
te Velthuis
Henk
H
Prakken
Berent J
BJ
Kuis
Wietse
W
Rijkers
Ger T
GT
D001171:Arthritis, Juvenile / Q000276:immunology; D001172:Arthritis, Rheumatoid / Q000276:immunology; D001773:Blood Cells; D016022:Case-Control Studies; D016207:Cytokines / Q000032:analysis*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology; D008214:Lymphocytes / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology; D010835:Phytohemagglutinins / Q000494:pharmacology; D040081:Protein Array Analysis / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D013745:Tetanus Toxoid / Q000494:pharmacology
D016207:Cytokines; D010835:Phytohemagglutinins; D013745:Tetanus Toxoid