Protein assays are indispensable, versatile tools with a myriad of applications across biochemistry, molecular biology, and biotechnology. In biopharmaceutical research, these assays are essential for studying cellular biology, exploring the relationship between protein structure and function, elucidating disease mechanisms, and informing the development of novel therapeutics.
In preclinical and clinical research, protein assays are commonly used for:
- Quantifying the concentration of proteins in a sample to support downstream analysis such as pharmacokinetics (PK)/absorption, distribution, metabolism, and excretion (ADME)
- Assessing the purity of isolated proteins, which is crucial in antibody preparation, enzyme isolation, and recombinant protein production
- Monitoring protein interactions with ligands or other moieties, which are fundamental to studying cellular processes, signal transduction pathways, and drug-target interactions
- Measuring enzyme activity, which is vital for studying metabolic pathways, drug metabolism, and the effects of inhibitors or activators on enzyme function
- Diagnosing and monitoring disease through the detection and quantification of biomarkers
There are multiple protein assay platforms, each with pros and cons. To select the most effective assay method for answering a particular scientific question, it is important to understand both the platforms available and the question being asked. This article will help inform your selection process.
Common protein assay platforms
Enzyme-linked immunosorbent assay (ELISA)
Technology and common applications: ELISA, the most traditional format for ligand binding assays, uses the catalytic properties of enzymes to detect and quantify immunologic interactions. The most common approach to using the ELISA technique involves detection of the presence of a ligand in a liquid sample using antibodies directed against the protein to be measured.[1] This technique is widely used for biomarker profiling.
Pros: There are a variety of commercially available kits, and no conjugation of antibody is needed, so the time needed to develop an assay is relatively short. Moreover, ELISA assays do not require a special plate reader for readout.
Cons: Compared to other technologies, ELISA has a small dynamic range (1-2 log), long assay duration, and large sample volume requirement (100 µL). In addition, it is not easy to multiplex. Thus, many ELISA assays have been converted to MesoScale Discovery (MSD) format.
Electrochemiluminescence (ECL)
Technology and common applications: ECL, using the instrument platform developed by Meso Scale Discovery (MSD), is a robust and widely-utilized technology for bioanalysis, including biomarker and cytokine profiling. This technology is also frequently used for PK and anti-drug antibody (ADA) assays, for both non-clinical and clinical studies.
Due to their multiplexing capability, MSD assays are commonly used for cytokine profiling or discovery and profiling of novel biomarkers in early-phase clinical trials. MSD provides both high performance, validated assays and easy-to-build personalized multiplex assays with up to 10 analytes. MSD offers the following platforms and kit types, which vary based on sensitivity, validation, and customization:
- S-PLEX is an assay platform with high sensitivity (fg/mL), well-suited for low concentration biomarkers
- V-PLEX offers ng/ml sensitivity and analytically validated single-plex and multiplex kits, depending on context of use
- R-PLEX and U-PLEX are platforms that support customizable multiplex assays
MSD assays are widely used for studying biomarkers in serum, plasma, CSF, cell lysates, cell culture supernatants, urine, and tissue homogenates. MSD assays have even been successfully utilized to measure biomarker concentration in unusual matrices such as peripheral blood mononuclear cells (PBMCs), bronchoalveolar lavage (BAL), and fecal samples.
Pros: MSD has a wide dynamic range (3-4 logs, even up to 5 logs), low matrix effects, high sensitivity, low sample volume requirement (≥ 25uL), and flexible multiplex opportunities (up to 10-plex).
Cons: Currently, only MSD can conjugate the detection antibody with their proprietary TURBO-BOOST label, limiting the ability of researchers to develop assays in house. However, it is possible to use biotin and SULFO-TAG to label antibodies and develop an MSD-based assay from scratch or to convert an ELISA to an MSD assay to improve performance.
Olink
Technology and common applications: Olink is a newer technology for ligand binding assays. It is a powerful tool for early-phase biomarker profiling as an exploratory endpoint. Olink offers various panels, including:[2]
- Target 96 & 48 panels for targeted protein biomarker discovery
- Flex, a made-to-order product customized with up to 21 human proteins selected from ~200 pre-validated protein biomarkers
- Focus, a custom panel of up to 21 proteins from Olink’s Target and Explore libraries
Olink’s Proximity Extension Assay (PEA) technology with qPCR readout makes it possible to achieve very low sample volume (1 µL) due to signal amplification. Olink has published data files with validation characteristics for ~200 proteins, including limits of quantitation, range, and precision. These data files are useful for sensitivity comparisons when deciding which platform to pursue for protein profiling. In addition, Olink publishes procedures on how to process alternative matrices such as tissue samples, tissue lysates, dried blood spots, and more.
Pros: Olink offers high throughput (96- or 48-plex), wide dynamic range (3-4 log), high sensitivity, and very low sample volume requirement. Another advantage is its compatibility with a diversity of sample types from serum and plasma to dried blood spots, saliva, urine, fine needle biopsies, and other biological samples. The most significant advantage of this technology is its high specificity for multiplexed immunoassay since only matched DNA reporter pairs can hybridize to produce an amplicon for real-time qPCR so antibody cross reactivity will not be detected, thus enabling 96-plex.
Cons: Olink technology only measures samples with single replicate, and the software is not suitable for regulated assays as Olink is still working toward 21 CRF Part 11 compliance. Another disadvantage is that a minimum of 15-21 targets are required to use this technology, so any panel below 15 biomarkers will not be a good candidate for this technology.
Quanterix
Technology and common applications: First introduced in 2013, Quanterix is a technology that takes advantage of their proprietary Simoa® bead technology using paramagnetic particles and can detect protein at ultra-low concentrations. Quanterix can reach ultra-high sensitivity of fg/mL, has great dynamic range (>4 logs), and is widely used to detect CNS biomarkers (e.g., N4PA, N4PB, N4PE).
Simoa® assay kits span multiple therapeutic areas including cardiology, infectious disease, inflammation, neurology, and oncology and can be run on any of Quanterix’s three instruments: HD-X™ Automated Immunoassay Analyzer, SR-X™ Biomarker Detection System, and SP-X imaging and Analysis System™. Researchers also have the flexibility to develop custom Simoa bead assays.
Pros: The biggest advantage for this technology is the capability to detect biomarkers at a femtogram level. Quanterix can also multiplex up to 4 analytes with its SR-X™ biomarker detection system. This technology is widely-used in neurologic disease biomarker detection due to its high sensitivity.
Cons: Simoa kits are more costly than ELISA kits and the number of proteins that can be analyzed with this technology is limited. It has also been observed that the newer kits from Quanterix sometimes do not work well with the SR-X, especially with multiplex kits that were developed for the HD-X system but should theoretically work with the SR-X. Moreover, the detection instrument is expensive, can only be used for SIMOA, and requires specialized training to operate.
Luminex
Technology and common applications: Luminex utilizes xMAP® bead-based technology that uses microspheres with unique spectral addresses and can quantify protein with high throughput. xMAP is the world’s most used multiplexing technology and can simultaneously detect up to 500 targets in a single run. Luminex has comparable sensitivity (pg/mL) and dynamic range (3-4 logs) to MSD, with a sample volume requirement of 50 µl..
xMAP technology is an open multiplexing platform so Luminex multiplex kits and instruments can be procured from other manufacturers and partners including ThermoFisher Scientific and BioRad under license from Luminex.
Pros: Luminex can multiplex up to 50 analytes in one well.
Cons: Use of Luminex is limited to early-stage research. It cannot be used in late-phase clinical trials, likely due to assay performance and software that is not fully 21 CFR Part 11 compliant. Read time is relatively long, at ~20-40 minutes per 96-well plate and ~75 minutes per 384-well plate.
Jess Automated Western Blotting
Technology and common applications: Jess is an automated Western Blot system. It is a combination of traditional Western Blot with ELISA. Jess separates protein by size and provides quantitative results. It is a powerful tool to measure total and phospho protein in the same sample. This technology also offers higher sensitivity in some tissue samples which other platforms are unable to detect.
Biotechne, the manufacturer of Jess, does not offer assay kits, but they do offer assay modules and a Simple Western Assay Kit Builder that allows researchers to design their own kits.
Pros: Jess can produce fully analyzed results in as little as 3 hours. Other advantages include low sample volume (3 µL), picogram-level sensitivity, and 2-3 log dynamic range.
Cons: It is not easy to multiplex (up to 3 plex) and sample numbers per run are 13 or 25, much lower than 96-well plate assays.
Choosing the most effective protein assay method
Taking the time to consider the protein detection options available and understand which is most appropriate for your research objective, budget, and timeline is essential for selecting the right assay.
Many factors need to be taken into consideration when deciding which assay format to use, including:
- Sample type and volume. The type and volume of sample available for analysis may dictate, or narrow down, the most appropriate technologies.
- Data use. Will the data be used for an exploratory, primary, or secondary endpoint? Does the data need to be quantitative, semi-quantitative, or qualitative? This will inform the level of validation required for the research objective.
- Analyte type and number. What analyte(s) will be measured and what is their expected concentration range in the sample? This will help determine whether a multiplexing technology is required and what level of sensitivity and dynamic range is necessary.
- Evaluate the number of samples to be processed and how quickly results are needed. In some cases, the need for high throughput may require an acceptable sacrifice in sensitivity.
- Available in-house platforms and budget. What tools can be accessed and what is the budget? Some protein assays require dedicated instruments and instrument-specific kits that may be costly.
- Available reagents and controls. Depending on level of validation, antibodies or recombinant proteins may need to be custom made, requiring long lead times.
- Downstream assays. If the samples are to be used for further analyses, it is important to ensure that the components of the protein assay do not interfere with any downstream applications.
For most protein ligand binding assays, MSD is a suitable platform due to its overall performance and customization options, with S-PLEX and V-PLEX being the most robust. However, when there are many analytes of interest, as is often the case when identifying and selecting biomarkers, high multiplexing is advantageous and Olink Target 96 is a good option.
Key takeaway
With advancements in technology, researchers now have access to a multitude of protein assay platforms. Understanding how these technologies differ can help researchers select the most appropriate platform for their analyte(s) of interest and their scientific objectives.
At Precision for Medicine, we are well-versed in the protein assay platforms available and have extensive experience in designing, developing, and validating protein assays to support biomarker discovery and biomarker-driven development programs.
Learn more about Precision's cytokine and protein biomarker assay services >
References
[1] StatPearls. Enzyme Linked Immunosorbent Assay. Available at https://www.ncbi.nlm.nih.gov/books/NBK555922/.
[2] Olink. Available at https://olink.com/.
