Cytokine research is essential to better understand the immune system and learn how dysregulated cytokine signaling is involved in disease. By multiplexing cytokine detection, researchers can gain a broader picture of the condition being investigated, as well as conserve limited sample material. MILLIPLEX® assays, a range of bead-based multiplex immunoassays centered on Luminex® xMAP® technology, provide cytokine detection at pg/mL levels in a wide array of biological fluids. Recently, they have been successfully used for characterizing neuroinflammation during progressive stages of neurological disease.
A challenge for cytokine research lies in detecting analytes that are expressed at low concentrations, often into the picomolar range. In addition, detection must usually be performed in complex sample types, many of which contain large numbers of proteins that can interfere with the accuracy of analyte measurement. Further problems stem from the fact that cytokine expression can vary considerably between samples, which makes it challenging to establish what constitutes abnormal expression and how this might correlate with disease. Moreover, because cytokines regularly function together to produce a coordinated response, multiplexed detection is often desired.
One of the main ways in which cytokine research challenges are being addressed is through the development of novel immunoassay kits. As well as offering superior specificity and sensitivity compared to existing methods, such products promise compatibility with a broader range of sample types and an increased capacity for multiplexing. To ensure researchers can fully leverage these benefits and achieve more consistent results, some manufacturers offer optimized protocols for sample preparation and handling, along with an extensive selection of support products (e.g., premixed standards and controls) and detailed guidance for data analysis and troubleshooting.
MILLIPLEX® kits are centered on the well-known Luminex® xMAP® platform, whereby color-coded beads—each coated with a different target-specific antibody—are used for capturing analytes from solution. Once the analytes are bead-bound, a biotinylated detection antibody is introduced, followed by PE-conjugated streptavidin. The sample is then read on a dual-laser Luminex® instrument based on flow cytometric detection, where a 635 nm laser is used for bead identification and a 532 nm laser measures the PE-derived signal. More than 200 MILLIPLEX® multiplex kits are currently available, including over 50 products for immunology and cytokine detection.
MILLIPLEX® assays have been extensively verified for the typical sample types used by many researchers: serum, plasma, and cell/tissue culture supernatants. More recently, they have also been analyzed for use with human urine, tears, milk, saliva, and cerebrospinal fluid (CSF) with the MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A—a kit designed for simultaneous measurement of 48 different immune factors at pg/mL levels.
For these experiments, samples were incubated with the beads overnight at 4˚ C in a 96-well plate with shaking. The beads were then washed and incubated with the detection antibody (1 hour), followed by incubation with streptavidin-PE (30 minutes). Next, the assay was read on a Luminex® 200™ instrument and the data was analyzed using Belysa® Immunoassay Curve Fitting Software. While the analyte concentration was found to vary depending on the sample type, all analytes in the panel were detectable in at least one of the samples.
To demonstrate the applicability of MILLIPLEX® assays to a disease setting, CSF samples from individuals with mild cognitive impairment (MCI) and Alzheimer’s Disease (AD) were run alongside CSF samples from healthy controls. Distinct responses were seen across the different groups, with key observations including elevated interleukin-9 (IL-9) in MCI and AD CSF compared to CSF from healthy individuals, and elevated macrophage-derived chemokine (MDC) in MCI CSF. Likewise, when serum samples from healthy donors and AD patients were compared, several cytokines showed significant differences in their expression.
In addition to the advantages already mentioned, multiplexed cytokine detection offers several further benefits. These include reduced cost per data point compared to running a panel of single-analyte assays and increased opportunities for introducing high-throughput screening. Also, with commercial kits like MILLIPLEX® assays being subject to rigorous evaluation of key performance criteria prior to release—including specificity, sensitivity, cross-reactivity, dilution linearity, kit stability, and sample behavior to name but a few—researchers using these products can have greater confidence in their results.
To learn more about MILLIPLEX® assays and their applicability to cytokine research, visit SigmaAldrich.com/milliplex-immunology.