The Trouble with ELISA
Development of the enzyme-linked immunosorbent assay (ELISA) in the 1970s was a great leap forward in biomedical research. ELISA assays are so valuable that they are still used today in many fields, including antibody development and screening. Yet, challenges with ELISA assays make it hard to translate this usefulness into a high-throughput screening application. In this article, we discuss some of the current challenges of the ELISA assay, some alternatives for immunoassay (particularly flow cytometry), and some tools and technologies that can address the limitations of ELISA and other immunoassays.
Complex Assay Development Workflows
ELISA assay protocols traditionally have complex workflows that include several time-intensive wash steps. Most alternative immunoassays, by contrast, such as Meso Scale Discovery (MSD) Electrochemiluminescence assays, Luminex Bead Based assays, AlphaLISA, and flow-cytometry, have streamlined and collapsed protocols for easy plate preparation without wash steps. For example, protocols without multiple wash steps are possible with flow-cytometry and bead-based multiplexing because the bead takes up most of the optical path of the laser in the detection system. A single wash step is, therefore, sufficient to remove almost all the background signal from unbound detection antibody.
Single end-point assays
ELISA allows assay of only one analyte at a time and so is not conducive to the time-saving and reagent-saving multiplexing of target proteins. With ELISA, three separate assays are required to test three analytes; however, Electrochemiluminescence assays, AlphaLISA, and multiplexed assays using flow cytometry enables testing multiple analytes in the same assay. For instance, MSD Electrochemiluminescence assays allows multiplex panels of up to 10 analytes and Bead based Luminex assays allows mulitplex panels of up to 50 analytes simultaneously. With flow cytometry, multiplexing is possible because of encoding technology. As an example, beads can be encoded by labeling with different dyes, then differentially coated with the antigens of interest. Because of the different dye encoding, these beads can be mixed together, and detected based on their fluorescence intensity, allowing simultaneous evaluation of antibody binding to multiple soluble antigens in the same assay. Encoding also permits simultaneous assay of differential antibody binding to cell-surface expressed antigens by mixing together different cell lines encoded with different dyes.
With flow cytometry and multiplexed bead- or cell-based assay, therefore, only one assay need be run to test interactions among dozens of protein pairs. Ultimately, this ability to multiplex reduces the number of assays that must be run to screen a number of antibody candidates, thus enabling researchers to miniaturize their reagent and sample requirements across a candidate screening program.
ELISA is not suitable for high-throughput screening and analysis
Because of the complexity of ELISA protocols and ELISA’s limitation to evaluating one analyte at a time, it is not suitable to use in high-throughput screening applications. In contrast, most alternative immunoassays are more conducive to high-throughput screening. Using a high throughput flow-cytometry-based platform, for instance, with rapid sampling technology, reduced sample volume requirements, and ability to run multiplexed assays, allows fast generation of very large data sets. In just one example, researchers from Avacta Life Science compared productivity with ELISA to the Intellicyt® iQue platform for evaluating 768 clones and five target antigens. Using the Intellicyt® iQue platform took less than half the time (2.5 days vs. less than 1 day) and used less reagents and fewer consumables (antibody consumption per target screened 250 µg vs. 1.5 µg and plates used 120 96-well plates vs. two 384-well plates). Watch this webinar to learn more.
Most alternative immunoassays require smaller sample volumes than ELISA, including traditional flow cytometry; however, even traditional flow cytometers are still too slow for high-throughput screening. To maintain accuracy, even a high-speed cytometer cannot exceed a flow rate of a few thousand cells per second. Depending on the subpopulation of interest, therefore, high-speed flow cytometers may sort up to 106 cells per hour, but screening experiments often require more cells than this to produce usable data. Due to its patented system for rapid sampling with no dead volume and delivery of samples in an air-gap delimited stream, an advanced flow-cytometry-based platform, such as the Intellicyt® iQue3, is an improvement over both ELISA and traditional flow-cytometers because assays can be run using a reduced sample volume relative to ELISA and using far less time relative to traditional flow cytometry.
Even traditional ELISA produces a vast amount of data, which can create data analysis bottlenecks. Running multiplex assays on flow-cytometry-based screening platforms exponentially increases the amount of data produced. For instance, even on a small scale, producing standard curves for protein quantification, calculating dose response curves for EC50 and IC50 calculation, and determining which measurements in an experiment meet the cut-off criteria can take a great deal of time and effort when done manually. Even inputting the data into a third party software system for analysis may not save much time and effort and introduces the risk of error from data input mistakes.
With powerful analysis software, however, data analysis can be fast and efficient. The Intellicyt® iQue3 has an advantage here because of the integration of ForeCyt software that both runs the system and helps with data analysis. ForeCyt software helps with analysis bottlenecks with built-in standard curves for protein quantification and profile maps that help focus the data from multiparametric assays to relevant criteria for a specific question or set of questions. With these tools built-in, no third-party data analysis software is necessary to produce usable data.
Using a cell-based approach in an advanced flow cytometry-based platform gives results that are more biologically relevant than bead-based approaches, such as Luminex, Meso Scale, and AlphaLISA, because target proteins can be presented on the cell surface rather than on the well of a plate. In addition, compared to standard ELISA or even traditional flow-cytometry, using a high-throughput flow cytometry-based platform with powerful analysis software, gives researchers the ability to get more usable data in less time and using fewer resources. Taken together, these advantages make a high-throughput flow-cytometry-based platform superior for today’s high-throughput, multiparametric, content-intensive antibody discovery programs.