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

Abstract

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.Cytokines are soluble proteins that are secreted by cells of the immune system. These proteins can alter the behavior and properties of different cell types. Although cytokine functions are complex, cytokine profiles are highly relevant parameters of an immune response. Different cytokines possess biological overlapping functions, and they have the ability to regulate production of other cytokines. Therefore, analysis of the function of the complete set of cytokines expressed within microenvironments (e.g., a site of inflammation) is often of more value than analysis of a single isolated cytokine (13).Cytokines can be quantitated at various levels. mRNA can be detected by real-time PCR; intracellular proteins can be measured by fluorescence-activated cell sorter staining of permeabilized cells, and secreted cytokines can be quantified with bioassays, enzyme-linked immunosorbent assays (ELISAs), radioactive immunosorbent assays, and microarrays. Multiplex assays for detection of cytokines at the mRNA (6) and cellular levels (16, 18) are commonly used. However, these assays have one or more limitations, like the need for a large sample volume or detection of precursor proteins rather than native secreted proteins. In addition, these techniques are time-consuming and laborious.Recent advances concerning applications for the simultaneous detection of proteins have resulted in different particlebased flow cytometric assays. These assays have proven to be very useful in the simultaneous detection of cytokines in body fluids. Unfortunately, at present, either the number of different microspheres or the availability of predefined kits limits these assays (1, 3). The Bio-Plex system employing the Luminex multianalyte profiling technology (Lab-MAP) allows individual and multiplex analysis of up to 100 different analytes in a single microtiter well (20).Our laboratory focuses on immunoregulation and immunotherapy of children with autoimmune diseases-in particular, juvenile idiopathic arthritis (JIA). Sample volumes are relatively small due to our patient population. For a number of cytokines, ready-to-use beads are available, but not for the full spectrum. To overcome these limitations, we chose to develop and validate our own multiplex assays with the Bio-Plex system. With this assay, we were able to detect human cytokines in antigen-stimulated peripheral blood mononuclear cell (PBMC) culture supernatants from both autoimmune and healthy individuals. We showed that it is a reliable, fast, and reproducible technique with a sensitivity that is comparable to that of conventional ELISAs. MATERIALS AND METHODSCell isolation and cultures. Heparinized blood ...

PAN–DR‐Binding Hsp60 self epitopes induce an interleukin‐10–mediated immune response in rheumatoid arthritis

“…PBMCs from 6 patients with RA, being representative of the whole group of RA patients, were cultured with or without the various stimuli. After 72 hours in culture supernatants, the cells were harvested, and cytokine production was measured with a multiplex immunoassay (36,38). Based on the profile of proliferative responses and the corre- sponding data on the protective epitopes in the adjuvant-induced arthritis model (p1 having close homology with a protective epitope in adjuvant-induced arthritis), we decided to focus on peptide pairs p1/p2 and p3/p4 for these further studies.…”

“…The concentrations of the following soluble mediators were measured: interleukin-1␣ (IL-1␣), IL-1␤, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, IL-15, IL-17, IL-23, tumor necrosis factor ␣ (TNF␣), and interferon-␥ (IFN␥). The detection range for all cytokines was 1.2-5,000 pg/ml, except IL-8 and IL-23, which had a detection range of 2.4-10,000 pg/ml (38).…”

“…Culture supernatants were harvested after 72 hours and stored at Ϫ80°C until further analyzed. Extracellular cytokines and chemokines were measured using a multiplex immunoassay (Luminex, Austin, TX) as previously described (36)(37)(38). In summary, the antibodycoated microspheres were incubated for 60 minutes with standards or culture medium (25 l) in 96-well, 1.2-m filter plates (Millipore, Amsterdam, The Netherlands).…”

Chemokines as risk factors for type 2 diabetes: results from the MONICA/KORA Augsburg study, 1984–2002

“…Serum levels of MCP-1, IL-8 and IP-10 were measured by Luminex multiplex technology using a Luminex 100 analyser (Luminex Corporation, Austin, TX, USA) based on a previously published protocol [26]. Fluorescent xMAP COOH microspheres were purchased from Luminex Corporation.…”

Multiplex bead array assays: Performance evaluation and comparison of sensitivity to ELISA☆

“…Several assay developers offer kits for the multiplex detection for human, mouse, and rat analytes, many of which are shown in Table 1. Some recent applications with Luminex-based fluorescent microspheres have included cytokine quantitation (12), hormonal analysis (13), single nucleotide polymorphism genotyping (14), growth factors (15), and characterization of the molecular interactions of nuclear receptors (16).…”