Premier MSD Assay Service For PK, Immunogenicity (ADA/nAb) And Meso Scale Cytokine Multiplex Bioanalysis!

Meso Scale Discovery is a robust platform to measure molecules in complex biological samples. Meso Scale Discovery assays can help you profile biomolecules such as intracellular signaling proteins and cytokines. Meso Scale Discovery combines electrochemiluminescence detection with multi-array technology to provide unsurpassed sensitivity and multiplexing capacities, making it an ideal detection platform for drug discovery and development studies. The Meso Scale Discovery multiplex system offers a wide range of assay options, including MSD immunogenicity assay, MSD cytokine assays, MSD PK assay, MSD ADA assay, and MSD cell based assay.

With 20+ years of experience developing sensitive immunoassays, NorthEast BioLab is committed to providing robust Meso Scale Discovery multiplex assays for measuring analytes such as biomarkers, phosphoproteins, and cytokines in complex biological matrices. Moreover, we offer MSD assay development and MSD assay optimization services to support and accelerate your drug development timelines. As your assay partner, we help you develop robust MSD assay protocols for high-quality MSD analysis even in the most challenging situations.

Our MSD multiplex assays provide greater analytical performance and customer value. We design numerous MSD assays, such as MSD biomarker assay, MSD ADA assay, MSD cytokine analysis assay, MSD cell based assay, and MSD PK assay, based on a variety of pharmacological and biomedical needs. Additionally, our MSD multiplex ELISA assays eliminate technical issues associated with other platforms while offering superior reliability, higher accuracy and sensitivity, and faster workflows. Besides, Multi-Spot panels in MSD ELISA assays provide multiplexing capacities to analyze multiple analytes simultaneously.

A majority of Meso Scale ELISA assays are sandwich-based assays. MSD labs employ assays such as Meso Scale PK and Meso Scale cytokine multiplex assays to evaluate drug concentrations in biological samples. In MSD cytokine analysis, the MSD method can be processed conveniently through 96-well or 384-well microtiter plates. Let us now dive deep into the working of MSD assays such as Meso Scale pharmacokinetics assay and Meso Scale PK assay.

MSD has a proprietary platform for measuring analytes in complex study samples. This technology has assays such as MSD cytokine multiplex assays to profile cytokines and cell signaling molecules, having a direct impact on drug development initiatives. MSD cytokine analysis consists of two crucial components, electrochemiluminescence detection, and Multi-array technology.

Electrochemiluminescence detection provides enhanced sensitivity, a broader dynamic range, and convenient sample analysis. It employs electrochemiluminescent labels that generate light when stimulated under appropriate conditions. This reaction is the basis of all MSD assays to measure important biomedical molecules. Moreover, MSD assays are highly reliable and provide accurate data through MSD data analysis in a broad range of sample types.

Multi-array technology is the combination of electrochemiluminescence and multiple arrays. This union brings speed and highly dense biological information. When applied to multi-spot plates, this technology can precisely quantitate several analytes in a single assay volume. Multi-spot MSD plates have arrays within each well. Each well can have up to 10 spots. Hence, a 96-well or 384-well assay plate can analyze numerous study samples, saving precious biological samples and reagents.

Moreover, MSD has two reliable instruments in its arsenal, MESO SECTOR S 600MM and MESO QuickPlex SQ 120MM. Both these instruments offer robust performance and enhanced reliability. Besides, these instruments work on efficient software designed for analyzing high-quality study data.

Meso Scale Discovery (MSD) assay is well suited for analyzing a wide array of biomarkers. We can run over 500 pre-validated MSD biomarker assays for a wide range of therapeutic applications, including immunology/ inflammation (cytokines and chemokines), intercellular signaling, cardiology, toxicology, and metabolic disorders. Our scientists can perform these MSD assays for a single analyte or multiplex with up to nine other compatible meso scale discovery biomarker assays. You can find a representative list of available analytes for different species here. For illustration, the table below highlights validated MSD cytokine and Alzheimer’s disease panels that can be adapted to suit your research needs.

Meso Scale Discovery Assay is a bioanalysis platform that utilizes electrochemiluminescence, unlike the colorimetric or chemiluminescent reaction in ELISA, as a signal detection technique. MSD ECL Assays are superior to traditional ELISA in many aspects, even as the various viable assay versions are quite like different types of ELISA. MSD Quick Plex 120 instrument applies the robust and sensitive electrochemiluminescence technology to quantitate single and multiple target analyte. MSD Assays enable accurate determination of analytes in complex biological matrices with improved throughput in a cost-effective and timely manner. Specifically, we can use MSD to analyze many clinically proven biomarkers, perform PK/TK analysis, and carry out ADA immunogenicity testing in a broad range of sample types such as blood, serum, and tissue, etc.

MSD ECL allows simultaneous multiplexing of up to 10 different analytes within the same well. Thus, the MSD platform requires 50-fold less sample than an ELISA method. Below, please see other crucial benefits of the Meso Scale Discovery technology –

• Absolute quantitation, short processing time, and low sample requirement (~10µl)
  • High standardization from simple protocols resulting in superior inter-laboratory reproducibility
  • Less time/labor intensive for scalability to larger lots
  • Low sample volume from human, rodent (mice, rat) and non-rodent (NHP, dog) species
• Higher sensitivity, better dynamic range, and reduced signal-to-noise ratio
  • Low analyte levels in small sample volumes
  • High/low abundance analytes (normal vs. elevated) within the same sample given large dynamic range
  • Limited matrix effect in serum, plasma, tissue culture media (TCM), and various other complex biological matrices
• Ready-made single analyte and multiplex kits with excellent performance and lot-to-lot consistency
  • Fit-for-purpose MSD portfolio: 400+ MSD V-plex (pre-validated), U-plex (personalized multiplex), R-plex (not multiplexed), and S-plex (ultra-sensitive) assay
• Most suitable to detect low-affinity anti-drug-antibodies (ADA)
• Emission at ~620 nm solves any color quenching problems

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 assay fundamentally utilizes the principles of enzyme-linked immunosorbent assays (ELISA) to detect analytes. Thus, proper reagent labeling, material storage, equipment usage, protocol adherence, and assay technique are crucial for optimal results with all MSD ELISA assays. Applied to standard use cases, mesoscale discovery multiplex and singleplex assays come optimized for antigen-antibody binding and detection in various matrices. However, determining suitable diluents and dilution factors can become critical for detecting novel analyte combinations in untested biological matrices. Similarly, two technical aspects of an MSD assay’s success are not stacking the plates and shaking the plates at the correct speed for optimal binding. Reverse pipetting and pipetting to the bottom corner of each well is strongly recommended for all steps of an MSD assay to prevent the formation of air bubbles. These air bubbles may interfere with the light emitted from SULFO-TAG™ on the meso scale discovery platform that uses electrochemiluminescence to detect analyte concentrations. Our scientists can also develop custom MSD immunoassays to suit your research needs. For all MSD mesoscale ELISA assays, a matched antibody pair is essential to success. Generally, each MSD assay must get optimized for capture and SULFO-TAG detection antibody concentrations, incubation times, blocking and read buffers, and dilution factors.

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 recommend each microtiter plate contain at least a Seven (7) point calibrator/standard curve that encompasses the dynamic range of the Meso Scale Discovery electrochemiluminescence (MSD ECL) assay. Also, in MSD ELISA protocol, each MSD ELISA plate should include at least three positive controls and negative control with untreated matrix. At a minimum, we prepare three positive controls by serially diluting the highest concentration calibrator in your sample matrix to produce a high (≤70 % highest concentration), middle, and low (2-3 times lowest standard concentration) quality control sample in MSD ELISA protocol. Notably, our scientists analyze all MSD assay calibrator and quality control samples in duplicate on the Meso Scale Discovery platform to assess precision and accuracy. For MSD mesoscale multiplex assays, the calibrators and quality controls are established for each analyte quantitated. Our standard operating and quality control procedures ensure that each analyte’s MSD assay is robust and provides reliable results for your precious study samples.

We recommend preparing the calibration standards and samples at least in duplicate to evaluate possible variation and allow course correction for each Meso Scale Discovery (MSD) assay. After assessing your duplicated standards and samples on the meso scale discovery (MSD) platform, our scientists can be confident of assay performance by calculating various concentration summary parameters such as average, standard deviation, coefficient of variation (%CV), etc. According to GLP guidelines, the replicated samples should have a % CV concentration under 20%. Usually, these FDA guidelines ensure that the data obtained from your MSD ECL assay is precise. Here, the %CV gets calculated for each analyte in your MSD multiplex assay. Sometimes, our sponsors request sample analysis in triplicate when the chosen matrix contains a high level of interferents, or the assay precision is uncertain.

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.

Meso Scale Discovery platform uses Methodical Mind software for data acquisition and Discovery Workbench software for data processing and reporting. When the data collection is complete, our scientists analyze each analyte’s calibration curve for accuracy and precision of the meso scale discovery immunoassay through the MSD data analysis. We only share study results with the sponsor if the calibration curves and %CV from replicates are acceptable. Usually, our meso scale discovery electrochemiluminescence (MSD-ECL) assay reports include:

• Tables and Charts containing raw quantitation data
• Summary of immunoassay conditions
• Catalog and lot number of MSD kits

Furthermore, each assay’s raw data file includes concentration statistics, such as mean, standard deviation, and %CV for each calibration standard, quality control, and study sample. As needed, we can create a formal and audited hyperlinked PDF report. Our scientists are glad to help if you have any additional questions concerning documentation and reporting of your Meso Scale Discovery project results.