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Multiplexed Cytokine Analysis in Drug Development

Cytokines are proteins released by activated cells that serve as soluble messengers binding to target cells through surface receptors, signaling functional responses. The nature of cytokines is pleiotropic; a single cytokine can elicit varying responses in different cell types. Often, cytokines are produced as a part of a cascade wherein one cytokine causes the targeted cell to produce different cytokines that then go on to affect other cells to release cytokines, continuing the functional response. Cytokine signaling can result in either activation or inhibition of cellular function. 

Cytokine analysis can be a critical component of understanding both the safety and efficacy of novel drug therapies. Cytokines are commonly measured via immunoassays. A plate is coated with capture antibodies specific to the cytokine of interest. The sample is added to the plate allowing the cytokine to bind to the capture antibody. A labeled detection antibody is then added to the plate which also binds to the cytokine, creating a sandwich. This label can be measured on a plate reader system; the higher the signal, the more cytokine is present in the sample. 

Since cytokine effects are often multimodal in nature, multiplexing cytokine analysis to measure multiple cytokines simultaneously is desired, allowing for better understanding of potential cascades and interactions. As a result, numerous platforms have been developed that expand upon the singleplex immunoassay format to allow for multiplexed cytokine analysis. Multiplex analysis platforms include bead-based approaches such as the Luminex or flow cytometry bead arrays and electrochemiluminescence approaches such as MSD systems. Rather than using a single capture and detection antibody, these platforms use a specific combination of capture and detection antibodies to be able to measure multiple cytokines simultaneously. 

The Luminex platform utilizes the principle of flow cytometry to measure multiple cytokines via a bead-based system. Individual beads have unique fluorescent profiles, and capture antibodies specific for different cytokines are coupled to the different beads. Samples are added to these beads; if a cytokine is present, it will bind to the bead and be detected by a secondary antibody resulting in a spectral signature unique to that bead. This will read out as a positive signal for that particular cytokine on the Luminex system. 

Meso Scale Discovery (MSD) platform uses a similar principle to multiplex cytokine analysis. However, instead of using beads with multiplexed capability, the MSD system uses plates with specific detection antibodies spotted on different regions of the plate. If a cytokine is present, it will bind to the analyte and can be detected by the secondary antibody that is bound to a electrochemiluminescent tag resulting in a positive signal on the reader. 

Multiplexed cytokine analysis is a powerful bioanalytical tool, with the ability to measure as many as 40 different cytokines from a single sample. This form of advanced analysis requires technical expertise to not only operate the platforms but also interpret the data. Therefore, to maximize the success of a drug candidate, partnering with an organization that has expertise in multiplexed cytokine analysis is key. BioAgilytix offers both the MSD and Luminex platforms for multiplexed cytokine analysis and has extensive experience in measuring these complex signaling cascades. 


  1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785020/ 
  2. https://academic.oup.com/biomedgerontology/article/63/8/879/567391#9077982
  3. https://bmcimmunol.biomedcentral.com/articles/10.1186/1471-2172-10-52
  4. https://www.sciencedirect.com/science/article/abs/pii/S0022175908003116
  5. https://www.sciencedirect.com/science/article/pii/S0009898105004262

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