Cytokine Assay, Cytokine Quantification, Inflammatory Biomarkers

Challenge

Develop, optimize, and qualify multiplex immunoassay or cytokine assays for cytokine quantification to assess inflammatory changes of the subjects in different biological matrices utilizing tagged capture-antibodies and ruthenylated detection antibodies on a Meso-Scale Discovery (MSD) platform.

We performed this Cytokine Multiplex Analysis or Cytokine Assay And Fit-For-Purpose Immunoassay Qualification Using Meso Scale Discovery to simultaneously quantitate Eight(8) inflammatory biomarkers multiplex IL-1 beta/IL-1F2, IL-2, IL-6, IL-8/CXCL8, IL-10, IFN-alpha2a, IFN-gamma, TNF-alpha in Human Plasma.

Every step of this MSD ECL multiplex assay for cytokine quantification must be optimized to improve reproducibility and robustness.

Inflammatory Biomarkers - Standard Curves

Solution

One of our sponsors sought to determine the inflammatory status of the subjects in a clinical study following their drug administration. Here, we recommended a Meso Scale Discovery based eight (8) plex pro-inflammatory V-PLEX cytokine/chemokine panel.

This MSD platform-based cytokine assay utilizes antibody cocktails for the eight analytes in a 96-well plate layout to produce a reproducible assay with high sensitivity and specificity for these biomarkers. The Meso Scale Discovery platform’s inflammatory biomarker multiplexing capability allows the simultaneous quantification of multiple cytokines from a single sample, providing comprehensive insights into immune responses and inflammatory status. This approach is crucial in cytokine quantification assays, where assessing various inflammatory markers offers a holistic understanding of immune system dynamics and treatment efficacy.

The Meso Scale Discovery platform offers bioanalysis with improved sensitivity, limited matrix effects, and a broader dynamic range that enables the measurement of multiple analytes from a single sample (i.e., multiplexing). This capability is particularly valuable in cytokine assays. Furthermore, the MSD ECL platform offers stability and non-radioactivity with easily conjugated labels. These advantages make MSD a solid alternative to conventional colorimetric methods like ELISA for cytokine quantification.

Here, we coated the MSD multi-array cytokine assay plate for One hour at room temperature (RT) with a cocktail of capture-antibodies, enabling the detection of inflammatory biomarker multiplexes. Next, we prepared standard curves, quality controls, and samples as per FDA bioanalytical method validation and sample analysis regulatory requirements. Later, 1% BSA blocking, standards, QCs, and samples were added to the coated plate, and incubated at RT on a shaker. Afterward, we washed off the excess or unbound proteins in automated plate washers. For detection, our team added SULFO-TAG conjugated antibody and captured the ECL signal on an MSD imager.

Outcome

We developed and qualified a dependable multiplex cytokine assay on meso scale discovery for chemokines and cytokine quantification, serving as a valuable inflammatory biomarker multiplex. This cytokine assay has been meticulously validated and continues to support multiple projects with a keen interest in assessing inflammatory state.

Biomarker measurements are becoming increasingly important in understanding disease progression and the development of therapeutics. Cytokine assays are integral to this process, as cytokines are key mediators of inflammation and immune response. Cytokine quantification gives us valuable insights into disease progression and therapeutic efficacy. This underscores the significance of robust cytokine assays in advancing our understanding of various diseases and guiding the development of targeted therapeutics.

The traditional ELISA platform has long been valued for its effectiveness in single analyte quantitation. However, the emerging MSD platform presents a significant advancement in cytokine assays. With its ability to analyze multiple biomarkers simultaneously, the MSD platform revolutionizes cytokine quantification assays. This innovative technology offers unparalleled accuracy and precision, even with complex biological matrices. By harnessing the power of the MSD platform, researchers can achieve comprehensive cytokine quantification from minimal sample volumes. This capability enhances cytokine quantification studies’ efficiency and opens new avenues for understanding immune responses and inflammatory processes.