Meso Scale Discovery assay is an excellent technology for measuring analytes in life science and biomedical research. MSD cell-based assays have low detection limits, use minimal samples, provide linear dynamic range, and handle complex biological samples. At the core of MSD analysis are microplates that are specifically developed to offer unique benefits. Analytical laboratory services employ MSD ELISA assays for multiple applications such as immunogenicity testing and cytokine analysis. After initial method development, researchers can use Multi-spot plates for multiplexing and deliver efficient MSD data analysis.
MSD assays, such as Mesoscale cytokine multiplex assays, have a similar workflow to ELISA. The primary steps are coating MSD microplates with capture reagents, blocking and adding of calibrators and samples, adding detection reagents, and reading and analyzing data. The MSD ELISA protocol has several advantages over traditional ELISA. The most prominent benefit of MSD cytokine analysis is that it significantly conserves critical reagents such as proteins and captures antibodies compared to traditional ELISA assays. The current article is a step-by-step guide to the MSD ELISA protocol.
MSD ELISA protocol
MSD assays come in different formats with antibodies as the capture reagent. These assay formats include Sulfo-tag conjugated to detection antibody, biotinylated detection antibody binding to Sulfo-tag streptavidin complex, and detection antibody binding to Sulfo-tag conjugated anti-species antibody. Additionally, evaluations such as MSD cytokine multiplex assays can use other capture reagents such as cells, peptides, carbohydrates, kinase substrates, virus-like particles, and oligonucleotides.
MSD assays have two types of plate surfaces: high bind plates and standard plates. Standard plates have hydrophobic surfaces, while high-bind plates have hydrophilic surfaces. The amount of capture material to be coated on the microplate depends on a combination of surface type and electrode size. Following is a general workflow of the MSD ELISA protocol.
Step 1: coat the MSD microplate using solution coating or spot coating method and incubate based on the coating protocol.
Step 2: wash the plate and block the remaining wells using a blocking solution, typically with 0.5-5% MSD blocker A. Incubate the plates overnight or for 1 hour with shaking.
Step 3: wash the plates and add samples. Incubate for 1-2 hours at RT with shaking.
Step 4: wash the plates and add a detection reagent. Incubate at RT for 1-2 hours with shaking.
Step 5: wash the plates and add the read buffer. Analyze the plates using an MSD reader.
As mentioned above, plates can be spot-coated or solution-coated. While both methods are ideal for antibodies, spot coating provides more sensitivity. MSD recommends testing both techniques during assay optimization. High bind and standard plates use different coating buffers as they have distinct surface hydrophobicity. When using the spot coating method in standard plates, the coating buffer needs surfactant to spread and cover the entire working electrode. With the correct technique, the dielectric stops the fluid from flowing beyond the electrode. However, too much surfactant will result in the coating solution flowing beyond and towards the edge. This poor coating will lead to more assay variability.
In Conclusion
Meso Scale Discovery assays are a robust bioanalytical tool. However, adequate considerations are necessary while developing a working MSD ELISA protocol.
