Using Customized Multiplex Biomarker Assays on the MSD Platform

Biomarkers are measurable indicators of biological states or processes—including physiological parameters such as blood pressure and heart rate, as well as molecular biomarkers such as DNA, RNA, and proteins. Biomarker testing plays a critical role in disease diagnosis, patient stratification, risk prediction, treatment selection, and therapeutic monitoring. Among protein biomarkers, cytokines are widely recognized as important indicators of immune activity, inflammation, and disease progression, making them valuable targets for biomarker analysis in drug development and clinical research.

Meso Scale Discovery, Biomarkers and You

The Meso Scale Discovery (MSD) platform is a high-sensitivity, multiplexed immunoassay technology used for protein quantification and biomarker detection in drug development. It uses electrochemiluminescence (ECL) detection triggered by electrical charges from carbon electrodes on specialized plates, enabling rapid testing of multiple analytes in low-volume samples. It is considered a faster, more sensitive, and higher-dynamic-range platform. The multiplexing capabilities enable simultaneous measurement of multiple cytokines, enabling faster, cheaper analysis of many analytes.

MSD provides a broad portfolio of cytokine testing solutions based on its proprietary ECL technology and offers extensive experience in the development of multiplex immunoassays. MSD offers a wide range of ready-to-use kits that include all necessary reagents and buffers. These kits include a fixed set of cytokines for testing, with the cytokines provided as standards along with procedures for their detection. If a custom set of cytokines is needed, MSD provides the means for users to develop custom assays.

Custom assays can be developed using different MSD product lines, such as U-Plex and R-Plex reagents. After selecting the final biomarker panel, researchers should identify the availability of reagents for each cytokine of interest. If all reagents are available as U-Plex reagents, the next section can be skipped. If some reagents are R-Plex or none are available, and in-house reagents must be used, follow the steps in the next section below.

Assessing Multiplex Compatibility

MSD has evaluated all U-Plex reagents and confirmed that they can be multiplexed in a variety of combinations. However, when non-U-plex reagents need to be multiplexed with U-plex reagents, reagent compatibility needs to be evaluated. For this evaluation, calibration curves for each non-U-plex reagent or in-house reagent should be run alone and then combined with all other cytokine calibration curves on a single U-Plex development plate. Plot the single and multiplex curves on the same graph for each cytokine. If the curves are similar and demonstrate reproducibility and parallelism (i.e., the two lines are consistently overlapping), the non-U-plex reagents can be multiplexed with the U-Plex reagents.

Graph showing a calibration curve prepared and run by itself, and the same calibration curve prepared alongside other analytes’ calibration curves and run in a multiplex assay. In this example, the two curves demonstrate good parallelism, as they overlap and don’t diverge until very low concentrations.

If the curves are not comparable, different MSD assay diluents used to dilute calibrators and samples should be tested. Consult MSD technical services for suggestions on which MSD diluents to use.

Establishing Controls

The next step is to prepare control samples to monitor and accept assay performance. MSD does not provide premade controls for all kits, so the user must prepare them from the calibrator stock. As an example, first reconstitute one vial of calibrator in 250 μL of diluent. To prepare the high control, transfer 200 μL of reconstituted calibrator stock into 1800 μL of diluent. The middle control is prepared by adding 400 μL of the high control to 1600 μL of diluent. The low control is prepared by adding 400 μL of the middle control to 1600 μL of diluent. The controls are then aliquoted and frozen. The volumes indicated can be adjusted as needed. The controls should be analyzed and tracked over the course of multiple assays to evaluate accuracy and precision.

Evaluating Matrix Effect and Minimum Required Dilution (MRD)

Another set of critical parameters to evaluate early on is the Minimum Required Dilution (MRD) and Matrix Effect. Matrix Effect refers to any interference in the quantification of the desired analyte due to the other components in the biological matrix. MRD is the lowest dilution to mitigate this matrix effect. Obtain a pooled matrix sample, preferably one similar to those expected from the clinical trial (i.e., disease state). Perform a serial dilution of the matrix in assay diluent (e.g., 1:2, 1:4, 1:8, etc.). Then perform the assay. Matrix effect is likely present if the average calculated concentration, after dilution factor is taken into account, is lower for less diluted samples compared to more diluted samples. If no matrix effect is observed, then the average calculated concentration for all diluted samples should return more-or-less the same value once dilution factor is taken into account.

Table: Example of Matrix Dilution Evaluation Experiment

As shown in the data above, cytokine concentration remains relatively constant across dilutions (1:2 to 1:64), indicating minimal matrix effect. The MRD for this example assay would be 1:2.

If available, it is advisable to also perform dilution experiments on actual study samples in addition to vendor-provided pooled matrix material. Depending on the disease being studied, pooled matrix material for the relevant disease state may not be commercially available. Study samples are often more representative of the intended testing population and may contain disease-related factors, endogenous binding proteins, elevated inflammatory markers, hemolysis, lipemia, or other matrix components that are not fully captured in a generic pooled matrix.

In addition, analyte concentrations in pooled matrix material may be very different from actual study samples. These differences between pooled matrix material and actual study samples can influence analyte recovery, signal behavior, and dilutional parallelism and may ultimately affect the selected MRD for the assay. Evaluating dilution performance in representative study samples, therefore, provides greater confidence that the assay will perform consistently under real testing conditions and supports the selection of a dilution strategy that is both scientifically sound and operationally practical.

Taking a Holistic Approach to Multiplex Assay Development

When evaluating the parameters discussed so far, including background signal, matrix effect, minimum required dilution (MRD), and control performance, it is important to assess them collectively across the full cytokine panel rather than on an analyte-by-analyte basis.

Because multiplex assays measure multiple cytokines simultaneously, assay performance should be interpreted holistically, balancing sensitivity, reproducibility, dynamic range, and matrix tolerance across all analytes within the panel.

Additional Parameters

To comprehensively complete assay development, it is recommended to evaluate the following additional parameters. However, due to the variable nature of biological responses and the complexity of multiplexing, some parameters may be difficult or impossible to evaluate, and should be determined in a scientifically sound, case-by-case basis.

1) Accuracy and Precision

Accuracy and precision are performance characteristics that indicate whether the assay produces reliable, reproducible quantitative results for the analyte being measured. They are evaluated using the known-concentration QC samples, which are analyzed and then compared with their nominal values. Prepare QC samples spanning the assay’s quantifiable range. Perform the evaluation 3 times over multiple days. Include a second analyst performing the assay to provide a better assessment of accuracy and precision.

2) Specificity and Selectivity

Specificity is the ability of an assay to detect and quantify the analyte in the presence of structurally related compounds. A lack of specificity typically leads to false positives or overestimation of analyte concentration because the assay cross‑reacts with similar molecules. Selectivity is the ability of an assay to measure the analyte in the presence of unrelated components in the sample matrix. This includes matrix components (lipids, hemoglobin, rheumatoid factor, etc.), concomitant medications that are not structurally related, and other endogenous substances that may interfere. Specificity ensures the assay is truly measuring the intended analyte. Selectivity ensures the assay works reliably across diverse patient samples and clinical conditions.

3) Dilutional Linearity

Dilutional linearity demonstrates that samples with analyte concentrations above the assay’s quantification range can be diluted into the quantifiable range and still yield accurate, proportional results. This is typically assessed by looking at the specific analyte at concentrations above upper limit of quantitation (ULOQ) (usually through spiking studies) and diluting to within the quantification range.

It confirms that the assay response remains linear and reliable after dilution, indicating that the assay is not affected by matrix‑dependent nonlinearities, binding artifacts, or saturation effects.

Depending on the analyte and matrix, dilutional linearity of the analyte may not be possible (for example, in cases where the endogenous analyte concentration is very high in pooled, normal matrix).

In Conclusion

The MSD platform provides a powerful and flexible approach for custom multiplex biomarker assay development, enabling simultaneous quantification of multiple cytokines and protein biomarkers from limited sample volumes. Through careful assessment of multiplex compatibility, control performance, matrix effect, minimum required dilution, and other assay characteristics, researchers can develop robust multiplex immunoassays tailored to their specific biomarker testing needs.

At Emery Pharma, we have extensive experience developing custom multiplex biomarker assays on the MSD platform for drug development and clinical research applications. Our scientists work closely with clients to design, optimize, and implement cytokine testing strategies that generate reliable, high-quality biomarker data. Whether you need a custom cytokine panel or support with multiplex assay development, our team can help advance your biomarker program!

Originally authored by Yero Espinoza. This article was reviewed by Dr. Janet Liu, current Director of Biology.