A better understanding of the cellular and cytokine processes has led to the advent of newer therapeutics in numerous diseases. These therapeutics are capable of inducing downstream changes in the cytokine environment. Cytokines are small signaling glycoproteins capable of regulating the immune response. These are cell-mediated responses that can effectively modify the quality and vigor of an immune response. However, the intensity of a cytokine response depends on the type of cytokine involved, its availability, the interaction with complementary receptors, and downstream signaling. As cytokine reflects a particular inflammatory response, they are widely used as biomarkers in drug discovery and development.
But what is the drug development process? A drug development process involves identifying and validating potential therapeutic compounds in a controlled and regulated environment. Today cytokine analysis is used throughout the entire process, right from early-stage drug development to post-marketing analysis. However, as cytokines work in a network, analyzing multiple cytokines is necessary to better understand the underlying principles of disease or healthy states. Hence, the current article focuses on the simultaneous analysis of blood cytokines using multiplex immunoassays.
Multiplex analysis of blood cytokines
Limited sample volume has always been a major limiting factor in bioanalysis. However, multiplex immunoassays help overcome this limitation and comprehensively depict disease mechanisms or states. Although ELISA is the gold standard for quantifying cytokines in blood samples, multiplexing extends the quantification of several cytokines in the same blood sample. Several multiplexing approaches available, including electrochemiluminescence systems such as MSD and bead-based assays such as Luminex cytokine assays. Following, we discuss the working of Luminex analysis for detecting blood cytokines.
A typical Luminex cytokine assay is a sandwich immunoassay. The working is similar to a sandwich ELISA, except coating the capture antibody to the assay well, Luminex assay coats capture antibodies onto beads in a suspension. This capture antibody is specific to a single cytokine. Beads are labeled internally with a distinct proportion of fluorescent dyes. Hence, researchers can combine various beads and simultaneously quantitate up to 100 different cytokines in a single sample.
Once beads are incubated with the sample, a washing step removes any unbound protein. A biotinylated detection antibody specific to the cytokine of interest and a Streptavidin-Phycoerythrin label is added to the sample. For analysis, a flow cytometer-like machine is used to assess each well. These Luminex systems have a laser that simultaneously identifies distinct fluorescence signatures and quantifies the Phycoerythrin signal, corresponding to the amount of cytokine captured by the beads. The intensity of a Phycoerythrin signal is plotted on the standard curve to derive the quantity of cytokine on each unique bead set.
The above protocol is almost similar to other multiplexing methods, which follow the sandwich immunoassay principle and use a capture and detection labeled-antibody. Moreover, along with multiplexing functionality, Luminex also provides custom Luminex assay options for designing and developing tailored assay solutions.
Conclusion
Luminex analysis plays a crucial role in the drug discovery and development process. Sponsors are increasingly relying on cytokine measurements while designing a clinical trial. Hence, they should consider each aspect of cytokine analysis that may affect its reliability and reproducibility while evaluating cytokines in blood samples.