MSD: ECL Multiplex Assays For Cytokine ELISA, Immunogenicity, And PK
Meso Scale Discovery Electrochemiluminescence Assays (ECL assays) are an excellent singleplex and multiplex platform for assessing target analytes in complex biological samples. The platform offers ultra-low analyte detection, provides a broader dynamic range, and handles complex plasma, urine, cell lysate, and tissue homogenate matrices while using minimal sample volume. Thus, ECL assays offer one of the best commercially available immunoassay platforms for Cytokine ELISA, pharmacokinetics, immunogenicity testing. Here, ultra sensitivity multiplex assays allow measuring of multiple low abundance cytokines or biomarkers simultaneously and a broader dynamic range helps precisely estimate the concentration of these analytes with minimal dilutions. Similarly, Meso Scale Discovery ECL Assays offer an excellent platform for high sensitivity pharmacokinetics and immunogenicity assessments. Therefore, our veteran scientists at NorthEast BioLab rely on the Meso Scale Discovery ECL Assays for your crucial cytokine ELISA multiplex assays, Immunogenicity (ADA and nAb), and Pharmacokinetics (PK) applications when feasible.
Meso Scale Discovery ECL Assay Technology
ECL technology is a proprietary platform from Meso Scale Discovery to measure large molecules such as proteins, antibodies, nucleotides, etc. in complex biological matrices. ECL or MSD technology can profile multiple analytes, such as chemokines and cytokines, to aid drug development and research programs. ECL Assays combine electrochemiluminescence and multi-array technology to provide high sensitivity immunoassay multiplexing capacities. That said, ECL technology still leverages an assay protocol and workflow similar to ELISA with the following main steps:
Coating microplates with a capture reagent
Blocking all plate wells to minimize nonspecific binding
Adding samples and calibrators
Adding detection reagent
Reading the microplate
Analyzing results
1. Electrochemiluminescence Technology
Electrochemiluminescent assays provide a broader dynamic range, enhanced sensitivity, and feasibility. ECL technology can generate reliable and high-quality results in numerous sample matrices such as serum, plasma, and saliva, making it a robust tool for all bioanalysis.
Figure 1. A top-view image of a well in the MSD MULTI-ARRAY plate depicting the electrochemiluminescence reaction and the working electrode, counter electrode, and dielectric.
2. Multi-array Technology
Multi-array technology integrates ECL assays and multiple arrays to facilitate faster and highly dense biological assays. When applied to ECL multi-spot plates, multi-array technology can precisely quantify numerous analytes in a single assay volume. Hence, fewer samples and less time are required for bioanalysis.
Figure 2. MSD plates with different multiplexing wells and single wells.
Advantages of ECL Over Traditional ELISA
Meso Scale Discovery ECL analysis has several advantages over traditional ELISA assays. Meso Scale Discovery designs numerous assay types for all pharmacological and biomedical applications. Moreover, MSD immunoassay technology produces faster and superior results while maintaining simpler assay workflows.
Benefits of Meso Scale Discovery or ECL assays over ELISA.
Similar to ELISA assays, ECL also has many assay variations, including sandwich, bridging, direct, and indirect assays. Let us understand each of these assay types.
Sandwich ECL assay
In sandwich ECL assay, the microplates are coated with a capture antibody. This step is followed by adding the antigen and a secondary antibody conjugated to an enzyme. The conjugated complex gives a chemiluminescent indication in the presence of the substrate. The intensity of the ECL signal is directly related to the amount of antigen present in the sample.
Figure 3. Sandwich ELISA versus Sandwich MSD.
Direct ECL assay
In these ECL or Meso Scale assay formats, the antigen binds to the electrode and the primary antibody conjugated with the Ru metal ion detects the presence of an antigen.
Figure 4. Diagrams of direct and indirect MSD assays.
Indirect ECL assay
Indirect assays are similar to direct ECL format, but an additional secondary antibody binds to the primary antibody to detect the microplate-bound antigen.
Figure 5. Diagrams of direct and indirect MSD assays.
Bridging assay
These assays are used primarily when the target molecule is another antibody. Bridging assays are a particular type of sandwich ELISA, where a bridge is created between a labeled and unlabeled antigen to detect anti drug antibody (ADA) in the sample. These assays are beneficial for confirming antibody production against an antibody-drug conjugate or therapeutic antibody and are frequently used in ADA and PK assays.
MSD Multiplex assays are one of the most prominent features of ECL platforms. ECL multiplex assays capture different antigens at distinct spots within a single microplate well. The CCD camera in ECL multiplex systems can identify up to 10 spots within a single well. Thus, ECL assay services can simultaneously quantify up to ten different analytes using the same study sample.
Besides, there are several advantages of MSD multiplex assays. They can quantify multiple analytes in a single assay volume. This quantification, in turn, will require lesser sample volumes, saving crucial biological samples.
Meso Scale Discovery offers two main multiplex assay products; V-Plex and U-Plex. V-Plex is a pre-validated microplate bound with capture antibodies. On the other hand, U-Plex allows end-users to choose the primary antibodies of their choice, thus providing a tailored multiplexing panel for sample analysis. Hence, many companies prefer ELISA conversion to multiplex ECL assay for analyzing their preclinical or clinical samples given these advantages.
Numerous variations in the ECL platform have enabled the development of different assay formats, such as ECL PK assay and ECL cytokine assay. Moreover, each assay format can be performed in different ways. Some of the assay formats for sandwich ECL assay are discussed below.
Figure 6. Immunoassay formats using capture antibodies on MSD MULTI-ARRAY plates.
Typical Immunoassay
There are three different sandwich ECL assay formats possible.
Biotinylated detection antibody is bound to the Sulfo-Tag Streptavidin complex
The Sulfo-Tag complex is conjugated to the detection antibody
The detection antibody is bound to the Sulfo-Tag conjugate anti-species antibody.
Immunoassays Using Non-Antibody Capture Regents
Besides antibody capture reagents, ECL assay can immobilize carbohydrates, cells, lysates, virus-like particles, kinase substrates, and oligonucleotides on microwell plates.
Figure 7. Examples of immunoassay formats using nonantibody molecules as capture reagents on MSD MULTI-ARRAY plates. In addition to sandwich immunoassays, other formats, such as direct binding or competitive assays, are also feasible.
Electrochemiluminescence immunoassay (ECL immunoassay) plates have carbon electrodes at the bottom of wells to support ECL detection in assay formats.
ECL Assay Plate Surfaces
ECL assay plate surface comes in two types; standard plates with a hydrophobic surface and high bind plates with a hydrophilic surface. The amount of capture reagents to be coated depends on the combination of surface type and working electrode size.
Meso Scale Discovery recommends coating the microplate with specific concentrations for reproducible results across different plate lots. Furthermore, they provide recommended antibody concentration ranges for spot coating and solution coating along with the product. The table below gives an overview of the binding capacity of standard and high-bind plate surfaces. This data was obtained from Meso Scale Dicovery using Sulfo-Tag conjugated A/G reporter protein and IgG as the capture reagent.
Plate type
96-well, 1 spot
96-well, small spot
384-well, 1 spot
Standard plate
1.0 pmol/well
0.25 pmol/well
0.2 pmol/well
High bind plate
5.0 pmol/well
1.25 pmol/well
1.0 pmol/well
Generally, high bind plates are suitable for measuring analytes at higher concentrations. On the other hand, standard plates offer higher sensitivity but exhibit lower nonspecific binding. Hence, it is recommended to test both assay surface types to identify an optimal plate surface.
Researchers can spot-coat or solution-coat the Meso Scale Discovery ECL plates. Although both methods are feasible for antibodies, spot coating may provide more sensitivity. This increased sensitivity from spot coating results from limiting the signal to working electrode.
Solution Coating of Antibodies
In solution coating, researchers must dispense the antibody solution in the bottom corner of the well. Touching the sides of wells or pipetting without submerging the tip can increase assay variability.
Figure 8. Appropriate solution-coating technique.
Spot Coating of Antibodies
High bind and standard plates use different coating buffers. During spot coating of antibodies, standard plate buffers may require using a limited surfactant to cover the working electrode.
Figure 9. Appropriate spot-coating technique. The droplet of diluted antibody should be confined to the working electrode surface and not breach the dielectric barrier
Assay signal in High Bind Plates
This example compares the signal of spot-coated antibodies and solution-coated antibodies for four mouse antibodies against human cytokines. The data suggest that spot-coated high bind plates demonstrate a 2 to 3-fold higher signal than solution-coated plates. This data also means spot coating immobilizes more antibodies than solution coating.
Figure 10. Signals from solution-coated (A) and spot-coated (B) antibodies on High Bind plates are compared for four different mouse antibodies raised against human cytokines.
Figure 11. Comparison of solution-coated (blue) and spot-coated (red) capture antibodies shows that the spot-coated High Bind plates had 2- to 3-fold higher maximum signals compared to the solution-coated plates. This suggests that more antibodies can be immobilized through spot coating. Different antibodies have different signal profiles for solution vs. spot coating.
Assay signal in Standard Plates
This example follows the same procedure mentioned above, but instead of high bind plates, it uses standard plates for spot coating and solution coating of antibodies. Again, spot-coated standard plates demonstrated a higher signal compared to solution coating. Hence, whether it’s high bind plates or standard plates, spot coating is the ideal technique to immobilize the highest number of antibodies for analysis.
Figure 12. Signals from solution-coated (A) and spot-coated (B) antibodies on Standard plates are compared for four different mouse antibodies raised against human cytokines.
Figure 13. Comparison of solution-coated (blue) and spot-coated (red) capture antibodies shows that the spot-coated Standard plates had higher maximum signals compared to the solution-coated plates. This suggests that more antibody can be immobilized on the spot through spot coating. Different antibodies have different signal profiles for solution vs. spot coating.
ECL Assay Plate Coating For Non-Antibody Molecules
Other than antibodies, ECL assay plates can immobilize additional molecules, such as peptides, antigens, carbohydrates, membranes, and oligonucleotides. However, different concentrations of coating buffers must be tested for non-antibody molecules. The choice of assay surface depends on the interplay between electrical charges on small molecules.
Researchers can immobilize cells and membranes through passive adsorption. This process directly binds the molecule without using binding agents. However, ECL recommends using gas-permeable seals for sealing the plates during coating. On the other hand, researchers can coat peptides directly or by conjugating them to a carrier protein. Generally, ECL recommends 5 to 10 ng/spot for small proteins and peptides. However, this volume must be varied depending on the binding capacity of the ECL plates.
Figure 14. Cell binding assay on MSD High Bind plates
Figure 15. Titration of mouse antiserum on MSD plates coated with different concentrations of a peptide used as the capture reagent.
Steps To Follow For Detailed Sandwich Immunoassay Protocol With ECL Plate Coating
Before starting the Meso Scale Discovery ECL assay, one must conjugate the detection antibody with a Sulfo-Tag. Either they can directly conjugate the primary antibody with the Sulfo-Tag molecule or introduce a secondary reporter molecule and couple it with an unlabeled detection reagent. Following is an example of a detailed sandwich immunoassay protocol with the ECL method.
Coat the ECL plate depending on the surface type
Block the plate if required
Prepare the assay controls and calibrators
Wash the plates and add assay controls and calibrators
Prepare the detection antibody
Wash the plates and add the detection antibody solution
Prepare the read buffer
Wash the assay plates and read them
Figure 16. Schematic diagram of the typical steps in an MSD assay.
Researchers can store uncoated ECL assay plates at room temperature. However, once the plate package is opened, it is recommended to keep the unused plate in the original packaging. Unopened plate packages have a 30-month shelf life.
Steps To Follow For Typical ECL Plates Layouts For Assay Optimization
Researchers can consider several steps to optimize ECL assays. They must test multiple titrations for detection and capture antibodies to increase assay signal and reduce background noise. Besides, the maximum assay signal must be less than 1 million counts.
If a high background is present without the sample, one can use lower antibody concentrations. Users must vary the incubation times. Moreover, analysts must test different diluents for optimal dilution linearity and recovery in different study matrices.
Meso Scale or ECL technology has numerous applications as a highly advanced bioanalytical testing tool. From ECL or MSD cytokine panels and cytokine ELISA formats to cardiac biomarkers and ubiquitinated proteins, ECL assays are offered by Meso Scale Discovery through several products.
NorthEast BioLab offers bioanalysis services on highly sensitive ECL assay platform for preclinical and clinical sample analysis, including cytokine, PK/PD, and ADA assessments.
Meso Scale Discovery uses control samples covering the entire quantifiable range to provide absolute quantification across assay runs and lots. Besides, ECL has manufactured more than 400 V-Plex kits with robust analytical performance.
V-PLEX Assays Demonstrate a High Degree of Lot-to-Lot Reproducibility
Meso Scale Discovery uses control samples covering the entire quantifiable range to provide absolute quantification across assay runs and lots. Besides, ECL has manufactured more than 400 V-Plex kits with robust analytical performance.
V-PLEX Analytes
Meso Scale Discovery provides V-Plex analytes as part of the ECL multiplex panel or individual validated assay. These analytes are grouped into human, rat, mouse, non-human primate, and Human/NHP/Mouse/Rat/Canine.
Preconfigured V-PLEX Kits
Moreover, a subset of V-Plex analytes that meet all specifications for performance and quality is available in a preconfigured panel. They are available in 1,5 and 25-panel plates.
V-PLEX Assay Platform
V-Plex assays are validated assay kits. They offer a broader dynamic range, consistency, and higher sensitivity, making them ideal for many long-term studies.
ECL U-Plex assays allow researchers to build their immunoassays without any additional equipment. U-Plex ECL assays are highly flexible and can be personalized depending on individual assay needs.
U-PLEX Assay Development Workflow Procedure
The ECL U-Plex protocol consists of three basic steps:
Couple the biotinylated antibody and the U-Plex linker
Coat the microplate wells with specific linkers
Run personalized combinations of ECL U-Plex assays.
U-Plex custom assays provide tailored options to combine ECL assays with the analyte of your choice. Besides, U-Plex development packs allow custom multiplexing with up to 10 activated spots.
Today research suggests that diabetes, metabolic syndromes, and cardiovascular diseases have complex pathologies. These complex networks have increased the demand for robust biomarkers and stringent MSD U-plex protocols. Besides, obesity is linked directly to diabetes, metabolic syndrome, and hypertension. Hence, the ECL U-Plex platform has simplified these biomarkers in the following three groups.
U-PLEX Metabolic Human Biomarkers
U-PLEX Metabolic Mouse Biomarkers
U-PLEX Metabolic Rat Biomarkers.
Moreover, each of these group 1 assays is available on both Meso Scale multiplex and singleplex platforms.
Apart from the above metabolic combinations, ECL also has another comprehensive list of metabolic combinations. These combinations are also grouped into:
U-PLEX Metabolic Human Combinations
U-PLEX Metabolic Mouse Combinations
U-PLEX Metabolic Rat Combinations.
U-PLEX Assay Platform
U-Plex assays are personalized assay platforms. They provide more flexibility, allowing the use of specific reagents.
S-Plex is a ultrasensitive platform for detecting analytes at low femtogram levels. Their lower limit of detection is up to 1000-fold reduced compared to other assay methods.
The S-Plex procedure has three steps:
Assembling the immunoassay
Enhance, consists of adding the assay reagent to improve and generate the signal
The read step involves adding the ECL GOLD read buffer to measure the generated assay signal.
NorthEast BioLab provides S-Plex services to clients in need of highly sensitive assays. Meso Scale Discovery offers conjugation with either Sulfo-Tag or biotin as detector and capture reagents. These assays are available for a wide range of molecules, such as MSD mouse cytokine ELISA panels.
R-Plex assays are rapid and easy-to-design immunoassays with all advantages of the ECL and Multi-array technology. Mostly, R-Plex platforms offers highly versatile ECL immunoassays development formats using antibodies to nearly 400 analytes. Depending on singleplex or multiplex needs, researchers can choose their type of assay plates and combine them with R-Plex antibodies. The R-Plex assays work well for many research areas, such as ECL cytokine ELISA analysis, inflammation, cancer, cell signaling, immunology, and neurodegeneration.
The R-Plex antibody set consists of three major components:
T-Plex assay platforms are research-grade assay kits available for both single and multiplex assays. These ECL assays offer a wide range of multiplexed bioanalysis options with greater sensitivity, consistency, and dynamic range. These multiplex panels are grouped into Cytokine/Inflammation Panels, Metabolic Panels, Neurodegeneration Panels, Isotyping Panels, Toxicology/Injury Panels, Activated/Total Panels, and Intracellular Signaling Panels. Furthermore, they are compatible with multiple biological matrices. The following table compares the two main types of T-Plex formats.
ECL assay development tools are a suite of tools that meet all aspects of quality and performance. These tools include ECL GOLD products and the U-Plex assay platform. Furthermore, ECL assays offer ELISA conversion packs to transition existing ELISA assays to the ECL platform. These packs include assay formats such as sandwich, bridging, competitive and direct assays.
The ECL or MSD technology has two instruments at its disposal; MESO SECTOR S 600MM and MESO QuickPlex SQ 120MM.
Meso Sector S 600mm: The MESO SECTOR S 600MM is a flagship instrument. It provides rapid results, a wide dynamic range, and a low-noise CCD camera.
Meso Quickplex SQ 120mm: The MESO QuickPlex SQ 120MM is a versatile instrument. It specializes in ease of use, reliability, and low maintenance costs.
Equipment and Assay Analysis Software: ECL instruments run on the Methodical Mind and Discover Workbench software. Both these softwares can manage databases, analyze data, and come with data integrity tools.
At NorthEast Bioanalytical Laboratory, we employ the latest technology to provide you with best-in-class bioanalysis.
