The Meso Scale Discovery (MSD) platform provides a faster alternative to traditional ELISAs for performing sandwich immunoassays. The Meso Scale Discovery MSD assay platform utilizes Ruthenium (II) tris-bipyridine-(4-methylsulfone) [Ru(bpy)3] that, once conjugated to the analyte, serves as the tracer in competitive assays. The Ru(bpy)3-based tag undergoes a rapid redox reaction that emits light in the presence of an applied voltage. Assays for several eicosanoids have been adapted to the MSD platform. An optimized MSD electrochemiluminescent assay can quantitate host cell proteins with a simple protocol with few wash steps and at low cost.
Unique advantages of using the MSD Assay:
- Capture: 10-100 fold increase in capture capacity compared to ELISA
- Dynamic Range: 3-4 log dynamic range vs. 1-1.5 log range for ELISA
- Sensitivity: Detection of analytes at sub-picogram levels
- Reduced Sample Volume: 5-25 ul volume vs. 50-100 ul for ELISA
- Speed: The entire assay can be performed in 3 hours
- Multiplex Capability: Simultaneous measurements for up to 10 analytes
There seem to be Top 10 lists for everything these days so why not have one as well for MSD assay prep and analysis.
- Buffer Preparation
It’s best to prepare a single common buffer throughout the assay that will be used to prepare all diluents and to wash the plates. Preparing such a base buffer will eliminate any chance of variation due to different buffer dilutions. It’s also helpful to test out different buffers if you are looking to improve assay performance, this can include different buffers and concentrations. All buffer manipulations should be performed on ice and it’s important to complete the lysis buffer immediately prior to use to ensure no variation in potency.
- Antibody Preparation
Once the proper antibodies have been identified for the assays, they must be combined in multiplex assays. It’s been recommended that users send their antibodies to vendors for immobilization on MULTI-SPOT plates which will allow them to be immobilized on Multi-Spot electrodes and labeled appropriately. Proper laboratory technique should be followed while preparing the antibodies to reduce any chance of cross contamination. This includes changing pipette tips between samples and ensuring proper samples are dispensed in each vile.
- Sample Preparation
When preparing the serum and plasma its import that all solid material is removed by centrifugation. Plasma prepared in heparin tubes commonly displays additional clotting following the thawing of the sample and this can removed with centrifugation. Avoid multiple freeze/thaw cycles for serum and plasma samples since this can cause additional solid material buildup. Centrifuge the lysates at greater than or equal to 10,000 x g for 10 minutes to clear the cell debris from the lysates.
- Storage Conditions
To ensure total quality control of the MSD assay, its import to perform stability studies on the storage conditions for your controls and samples. This will verify that your storage conditions are adequate enough and will not cause variation in your MSD assays. Temperature variation can cause chemical changes in the samples and controls. It’s important to incubate the cells at 37 degrees Celsius and inhibitors at 4 degrees Celsius.
- Shaking Plates
It’s important to shake the 96 well MSD plates since it has been found that this accelerates capture at the working electrode. Shaking the plates also allows for proper mixing of reagents in the wells and coating the plates. Be careful to not have the shaker on a high setting which can cause the reagents to spill out of wells and cross contaminate. Typically shaking the plates for a few minutes is enough to accelerate the reaction without sacrificing sensitivity. It’s also helpful if the plate shaker is in a cold room or refrigerator to keep the reaction at a cooler temperature to ensure reactivity.
- Proper pipetting
Bubbles in the fluid can interfere with reliable reading of the plate and it’s helpful to use reverse pipetting technique to insure bubbles are not created when dispensing the Read Buffer. Speed of pipetting can also create bubbles in the fluid, it’s crucial to slowly aspirate and dispense the samples into the wells. Also double check the volumes when pipetting to ensure there is no variation between dispenses of samples.
Intra-assay variability is the within-run variation that represents the repeatability of the assay under the same conditions. The total assay precision should be calculated to insure reproducibility of the MSD assay. There are software programs available that can help you quickly spot such variation in your data
- Assay Optimization
Optimizing the MSD assay will improve the results these includes the antibodies, MSD diluents and incubation times. Testing different plates may also improve your results such as using the standard plate rather than a high bind plate.
- Run the Calibrators in duplicate
Run the calibrators in duplicate to generate the standard curve. The standard curve is modeled using least squares fitting algorithms so the signals from samples with known levels of the analyte of interest can be used to calculate the concentration of analyte in the sample.
- Use weighting algorithm
It’s important to use analysis software that includes the 1/y^2 weighting function in addition to using the 4 parameter logistic (4PL) or the 5 parameter logistic (5PL) models. The weighting function is important because it provides a better fit of data over a wide dynamic range, particularly at the low end of the standard curve.
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Hitachi Solutions offers a multiplex quantitative analysis solution, MasterPlex QT, that does 4PL & 5PL curve-fitting with weighting. It is compatible with many of the multiplex platforms that are out there in the market including:
- Luminex 100/200/MAGPIX/FlexMAP 3D xMAP systems