Meso Scale Discovery (MSD) Assays for PK, ADA and Cytokines by PhDs at 20+ Year, FDA-Inspected Lab

MSD is an electrochemiluminescence immunoassay platform: capture on carbon electrode plates, detection by a SULFO-TAG label that emits light only when voltage is applied at the electrode surface. Because unbound label never contributes signal, MSD delivers lower background, wider dynamic range and better sensitivity than a conventional plate ELISA, and it multiplexes up to ten analytes from one small sample. Here it carries PK, tiered ADA and cytokine panels from preclinical through clinical.

A wide menu: over 500 pre-validated MSD assays span cytokines and chemokines, inflammation, cardiology, toxicology, metabolic and neurology panels, across human and the usual preclinical species. Panels run as single analytes or multiplexed up to ten compatible targets per well, and where no kit reaches your analyte, a custom MSD assay is developed from reagents up. Send the analyte list and we map it before any money is spent.

MSD immunogenicity assays rely on a bridging assay format to detect anti-drug antibodies. First, we prepare a master mix with optimized concentrations of both the biotinylated and SULFO-TAG™ labeled drug. Our scientists then incubate the master mix and ADA reference standard or preclinical/clinical samples with the anti-drug antibody in a 96-well polypropylene plate. Thus, the anti-drug antibody bridges together the capture and detection labeled drug in MSD immunogenicity assays. Afterward, we add the incubation mixture to a blocked streptavidin plate and repeat an incubation before washing it. At this time, we are ready to quantitate the ADAs by electrochemiluminescence using the using read buffer on the meso scale discovery (MSD) platform. Generally, we can detect even low-affinity anti-drug antibodies using MSD assay as the protocol only requires one wash step. Furthermore, meso scale discovery immunogenicity assays do not use any species-related secondary antibodies, expanding its utility from preclinical to clinical studies.

Meso Scale Discovery (MSD) immunoassay acceptance criteria are similar to that for other ligand based assays (LBA) as per i) FDA Bioanalytical Method Validation Guidance for Industry (May 2018) as well as ii) Immunogenicity Testing of Therapeutic Protein Products — Developing and Validating Assays for Anti-Drug Antibody Detection (Jan 2019). We rigorously evaluate the accuracy and precision for each analyte in the meso scale discovery multiplex panel. Our scientists strictly follow MSD immunoassay protocols to establish duplicated seven-point calibration curves and negative controls using provided materials. We can include an additional calibrator at the lower concentrations for increased sensitivity in a meso scale discovery assay. Here, the calibrator/standard curves are evaluated using a 4-parameter logistic model, and each standard/control must have a deviation ± 20% of the nominal value. Also, the coefficient of variation between replicates should be ± 20%. Similarly, our scientists can add three to six positive controls in the QC samples to evaluate spike recovery, dilution linearity, matrix effects, and process stability. Furthermore, each MSD Assay gets rigorously assessed for the run-to-run variability.

MSD biomarker and PK assays are based on the principles of traditional ELISA. Thus, MSD assay method validation must be fit for purpose (FFP) and follow the FDA Bioanalytical Method Validation Guidance for Industry (May 2018). Ahead of assay validation, we perform method development, optimization, and validation preparation with multiple lots of assay materials for an acceptable bioanalytical outcome. For each Meso Scale Discovery immunoassay or sponsor-developed custom MSD assay, method validation starts with verifying appropriate critical reagents, calibrators, and controls. Then, each Meso Scale Discovery multiplex assay gets evaluated for accuracy and precision. The accuracy is assessed by treating each Meso Scale Discovery standard as an unknown and back-calculating the concentration using the standard curve. According to regulatory guidelines, the calculated value’s ratio (recovery) to the expected value should be within ± 20% (±25% LLOQ and ULOQ) of the nominal value. The precision of each standard, calibrator, and study sample is based on the calculated coefficient of variation (%CV) and should be no more than ±20% (±25% LLOQ and ULOQ). As needed, we perform sensitivity, selectivity, specificity, matrix effects (linearity and spike recovery), inter and intra-day variation, and sample stability to validate your MSD multiplex assay further.

An essential part of the drug development on Meso Scale Discovery (MSD) or other platforms is identifying and characterizing immunogenicity by measuring Anti-Drug Antibodies (ANA). Thus, our scientists follow the latest FDA guideline, namely Immunogenicity Testing of Therapeutic Protein Products — Developing and Validating Assays for Anti-Drug Antibody Detection (Jan 2019) in your preclinical and clinical studies. These guidelines ensure that the assay parameters (i.e., accuracy, precision, drug tolerance, specificity, and selectivity) for the chosen MSD Immunogenicity Assay type are appropriate for the intended purpose. One critical step in the MSD Assay validation process is using clinical samples to determine the cutoff for screening and confirmatory assays. For ANA screening, our scientists suggest a cutoff to provide a 5% false-positive rate. This cutoff helps identify individuals that may develop an immune response to a therapeutic protein. We lean towards a cutoff for confirmatory Meso Scale Discovery (MSD) Electrochemiluminescence Assays that decreases false-positive samples that may arise from non-specific binding. In summary, NorthEast BioLab team strictly adheres to FDA recommended tiered immunogenicity testing strategy (screening, confirmation, and titration) for your MSD Immunogenicity Assay validation.

All Mesoscale Discovery Immunoassays are based on ECL technology and provide a broad dynamic range and accurate results from a minimal sample volume. The Meso Scale Discovery multiplex assays are offered in five typical assay formats depending on your needs and applications:

V-PLEX: These are the highest quality assay panels from MSD. They are validated according to fit-for-purpose principles and contain controls with acceptance criteria to monitor plate-to-plate variability. These assays are available as a single validated assay or as part of a multiplex panel. All V-PLEX panels, including MSD cytokine assays, have been validated for serum, plasma, urine, and cell culture supernatants for multiple species.

U-PLEX: This assay format is the most flexible and allows you to customize your own Meso Scale Discovery multiplex panels. You can create panels of up to ten (10) compatible analytes from a comprehensive MSD biomarker assays menu or with custom antibody pairs. Meso Scale Discovery U-PLEX immunoassays are available in multiple species and demonstrated to work on serum, plasma, and cell culture supernatants.

R-PLEX: These kits provide matched antibody pairs for building your own customized MSD assay. Each R-PLEX kit contains a biotinylated capture antibody, a detection antibody conjugated to a SULFO-TAG™, reference calibrators, and recommended diluents. Sometimes, it’s possible to perform these highly sensitive R-PLEX assays in an open spot on a U-PLEX panel as per your sample dilution requirements.

S-PLEX: This small group of MSD assays specializes in ultra-sensitive singleplex assays that can reduce the lower detection limit by 10-1000 fold. These assays involve using reagents that help enhance and generate the assay signal on the Meso Scale Discovery (MSD) platform.

T-PLEX: These MSD assays are ready to use single and disease-specific multiplex assays that include some of the most widely studied analytes. These Meso Scale Discovery multiplex panels are available for various species and are compatible with a broad range of sample types to study both secreted and intracellular biomarkers.

Meso Scale Discovery electrochemiluminescence platform offers two types of plates: SECTOR plates and QUICKPLEX plates. MSD ELISA assays developed on SECTOR plates are read by reading sectors of 4 wells at a time. These plates come in single spot wells for MSD single-plex assays and multi-spot wells (4, 7, or 10) for MSD multiplex assays. The spots at the bottom of each well consist of an electrode that binds to a specific capture reagent for each Meso Scale Discovery assay (MSD) assay. Our scientists can coat the plates with various capture molecules such as antibodies, antigens, peptides, and lysates. After coating, we add the sample and detection antibody conjugated to Meso Scale Discovery electrochemiluminescence tag (SULFO-TAG™) over a series of incubation and wash steps. Once the assay is complete and read buffer is added, MSD platform applies a voltage to the plate’s electrodes to activate the SULFO-TAG™ for detection. QUICKPLEX is a newer single spot plate design that reads only one well at a time, and that’s intended for the development of a custom MSD assay.

Furthermore, MSD ELISA plates are available in three different surface types: standard streptavidin, small spot streptavidin, and high bind avidin. Both Streptavidin plates consist of a hydrophobic surface that contains a binding capacity of up to 1.0 pg/mol per one 96 plate well. In comparison, high bind avidin plates consist of a hydrophilic surface that exhibits a binding capacity of up to 5.0 pg/mol per one 96 plate well. Generally, Streptavidin plates offer higher sensitivity, while high bind avidin plates allow the detection of higher concentrations of analytes. Small spot streptavidin plate provides the highest MSD ELISA assay sensitivity. We recommend testing all relevant microtiter plates depending upon identified coating concentration and assay sensitivity requirements during your MSD assay development and validation.

Meso Scale Discovery (MSD) platform is designed for easy method transfer of an existing ELISA protocol to MSD assay. Among other considerations, we need two alterations when transferring an ELISA to MSD ECL assay. The capture reagents (directly or indirectly through a biotin label) must bind a spot on the plate containing an electrode. The detection antibody must also include a SULFO-TAG™ that emits light when the electricity gets applied to the electrode. Once both antibodies are slightly modified to meet the outlined criteria, the assay conditions need to be optimized for capture antibody concentration, the volume of capture antibody per well, and detection antibody concentrations. After the MSD assay is optimized, our scientists will revalidate the assay with a fit-for-purpose approach for parameters such as accuracy, precision, sensitivity, matrix effects (linearity and spike recovery), and sample stability. At this time, our newly transferred MSD ELISA assay is ready for analysis of your study samples.

Meso Scale Discovery (MSD) Assay platform offers numerous advantages over traditional ELISA. Generally, MSD ELISA assays exhibit higher sensitivity and precision, a more comprehensive dynamic range (3-4 logs), and a lower background signal than a conventional ELISA. However, one significant advantage is ability to develop custom Meso Scale Discovery multiplex panels for quantitating up to 10 analytes per well using small sample volumes (~25 uL). Moreover, MSD mesoscale assays have a robust matrix tolerance. Consequently, our sponsors receive high-quality data from their various sample types, including serum, plasma, cell culture supernatant, and tissue lysates. In conclusion, MSD mesoscale assays are highly flexible and simplify workflows to enable biomarker and cytokine testing when sample volumes are limited.

MSD Electrochemiluminescence or electrogenerated chemiluminescence is the emission of light by electrochemical assay reactions. All Meso Scale Discovery electrochemiluminescence assays (MSD ECL assays) utilize 96 well plates that contain single spot or multiple spots on the bottom of each well. These spots have electrodes that bind to various capture molecules such as antibodies, antigens, peptides, and lysates. After the capture molecules are attached to the plate, the samples and detection antibody conjugated to an MSD electrochemiluminescence tag (SULFO-TAG™) are added over a series of incubation and wash steps. Once the MSD mesoscale assay is complete, the user adds a read buffer, and a voltage is applied to the plate electrodes to activate the SULFO-TAG™ for detection. Now, the emitted light intensity gets measured by the Meso Scale Discovery (MSD) platform to quantify various analyte concentrations in your study samples. Using this relatively new technology, MSD ELISA exhibits high sensitivity and a more comprehensive dynamic range than traditional methods.

Each singleplex and multiplex Meso Scale Discovery electrochemiluminescence assay (MSD ECL assay) is designed to have a simple assay protocol and efficient turnaround time. Most MSD assay protocols are optimized such that the user can complete the time from sample preparation to reading quantitative results in a typical workday. Although incubation times between diverse MSD ELISA assays can vary in different MSD ELISA protocols, these are carefully controlled and monitored to retain high inter-assay accuracy and precision. Typically, MSD assays have a higher sensitivity and a more comprehensive dynamic range to avoid multiple dilutions for multiplex assays, even as they take a similar amount of time when compared to a traditional ELISA. Furthermore, the MSD multiplex platform is advantageous as compared to multiplex bead-based methods. Notably, Meso Scale Discovery (MSD) platform takes only 1 1/2 minutes to analyze a 96 well plate, while bead-based assays can take up to 1 hour.

We can apply Meso Scale Discovery (MSD) assays to analyze biomarkers in various biological matrices such as blood plasma, serum, urine, CSF, saliva, synovial fluid, and cell lysates, etc. Additionally, our scientists can use MSD assays to test tissue lysates. Here, the lysate sample preparation varies by tissue type, and our experts may recommend certain protease inhibitors to avoid degradation. Before launching the usual meso scale assay protocol, we quantitate the total protein through a Bradford/BCA assay and dilute its concentration to 12.5 ug/mL per well using recommended buffers. That said, most V-PLEX meso scale discovery electrochemiluminescence assays have only been validated for serum, plasma, urine, and cell culture supernatant matrices.