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Clinical and Diagnostic Laboratory Immunology, January 2003, p. 133-139, Vol. 10, No. 1
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.1.133-139.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Simultaneous Detection of 15 Human Cytokines in a Single Sample of Stimulated Peripheral Blood Mononuclear Cells

Wilco de Jager,1 Henk te Velthuis,2 Berent J. Prakken,1 Wietse Kuis,1 and Ger T. Rijkers1*

Department of Pediatric Immunology, University Medical Center Utrecht, Wilhelmina Children's Hospital, 3584 EA Utrecht,1 Sanquin Research at Central Laboratory of The Netherlands Red Cross, Department of Reagents, 1066 CX Amsterdam, The Netherlands2

Received 11 March 2002/ Returned for modification 17 July 2002/ Accepted 22 August 2002

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 1{alpha} [IL-1{alpha}], IL-1ß, 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.

* Corresponding author. Mailing address: Department of Pediatric Immunology (KC03.068.0), University Medical Center Utrecht, Wilhelmina Children's Hospital, Lundlaan 6, 3584 EA Utrecht, The Netherlands. Phone: 31 (0) 30 250 4353. Fax: 31 (0) 30 250 5350. E-mail: g.rijkers{at}wkz.azu.nl.

Clinical and Diagnostic Laboratory Immunology, January 2003, p. 133-139, Vol. 10, No. 1
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.1.133-139.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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