Immunoassays have different capabilities depending on the format that is chosen. This editorial provides an overview of the most commonly used immunoassays, touches on their limitations, and highlights some newer alternatives.
Established techniques remain popular despite known limitations
If you check the “applications” page on any antibody manufacturer’s website, you will likely see western blot, ELISA, immunocytochemistry (ICC), immunohistochemistry (IHC), and flow cytometry listed. So, why are these techniques so popular, and will they continue to dominate scientific research?
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According to Derrick Campos, Product Manager for the Octet® SF3 at Sartorius, researchers have persevered with established methods because they are heavily entrenched, reasonably easy to perform, and use relatively inexpensive reagents. However, he has noticed a recent shift toward using newer technologies. “A main limitation of established techniques is that they only allow for end-point analysis,” he says. “They are also very hands-on, with little walk-away time, and often generate large amounts of biohazardous waste. While such methods get people the information they need, provided that is all they need, many researchers want more, and it is this that is driving the development of new technologies.”
Dominic Andrada, Senior Marketing Manager for Scientific Applications at Luminex Corporation, adds that further constraints of existing methods include limited capacity for multiplexing, low throughput, and poor reproducibility, depending on the chosen technique. “While automated sample preparation and liquid handling have helped to address some of these problems, method development and optimization make their implementation slow,” he says.
Table 1. Overview of common immunoassay formats, including typical sample type and readout
| Format | Sample type | Method | Readout | Advantages |
|---|
| Western blot | Cell lysates, tissue homogenates | Proteins are separated by polyacrylamide gel electrophoresis (PAGE) before being transferred to a nitrocellulose or PVDF membrane for antibody-based detection | Chemiluminescent or fluorescent | Requires only small amounts of sample, results are easily interpreted |
| ELISA | Cell lysates, tissue homogenates, culture supernatants, other biological samples (e.g., serum, urine, saliva) | An analyte in solution is captured on the wells of a microplate before being detected with antibodies; the most common assay format is the sandwich ELISA, which uses matched antibody pairs for analyte capture and detection | Chromogenic or chemiluminescent | High sensitivity, quantitative, flexible, compatible with automation |
| Immunocytochemistry (ICC) | Intact cells | Cells are immobilized on microplate wells or glass coverslips and subjected to antibody-based detection | Fluorescent | Provides spatial context, including details of which sub-cellular compart-ments express the analyte |
| Immunohistochemistry (IHC) | Formalin-fixed paraffin-embedded (FFPE) or frozen tissue sections | Excised tissue samples are sectioned with either a microtome (FFPE samples) or a cryostat (frozen samples) and stained with antibodies for targets of interest; signal amplification is sometimes performed with the avidin-biotin-complex (ABC) method or labeled-streptavidin biotin (LSAB) method | Chromogenic or fluorescent | Provides spatial context, including information about the relative abundance and co-localization of different proteins |
| Flow cytometry | Intact cells in suspension | The sample is stained with fluorophore-labeled antibodies, then the cells are directed in single file past an interrogation point where one or more lasers are focused; as the cells pass the interrogation point, they scatter the light and emit fluorescence; the resultant signals are detected by photomultiplier tubes (PMTs) and used to produce a graphical representation of the sample to allow for identification of specific cell populations | Fluorescent | Can measure multiple (20+) parameters on the same sample and rapidly collect information from millions of cells to allow for the identification of rare populations |
Traditional immunoassays require augmentation
Demand for advanced methods extends beyond scientific research and into patient care. “ELISAs and other antigen-specific immunoassays are healthcare workhorses, providing critical information to diagnose patients with infectious pathogens, autoimmune disease, and cancer,” reports Dr. John Shon, CTO at Serimmune. “Yet while these tests are quite good at diagnosing conditions, they are less adept at illuminating the big picture. COVID is an excellent example. Millions of people have relied on SARS-CoV-2 immunoassays to determine their status, and many let out sighs of relief when their tests came up negative, but people experiencing long-term symptoms still didn’t know what was causing them. To gather both broad and granular information, it is essential that we augment traditional immunoassays with next-generation solutions to understand the full spectrum of disease.”
Alternative technologies address known problems
Many different types of immunoassays have been developed to overcome challenges for established methods, including the following:
- Increased multiplexing capabilities with Luminex xMAP® Technology
xMAP assays use capture reagents coupled to beads, each of which has a unique spectral signature, to analyze up to 500 proteins (immunoassays) or genomic analytes in a single sample. Once each bead set has bound to its target, a fluorophore-labeled detection reagent is added to allow for analyte quantification with a dedicated Luminex instrument.
“What makes xMAP Technology special is that many labs have applied their ideas and assays on our beads,” says Andrada. “While Luminex has been around for almost 30 years, it’s the novel biomarkers and inventive assay formats on xMAP beads that provide versatility to address changing requirements in life science research, applied, and diagnostic applications. For example, Luminex has helped vaccine development efforts with measuring seroconversion and functional antibody responses from multi-valent HPV vaccines, as well as being used recently for SARS-CoV-2 vaccines. In addition, xMAP Technology has been applied for measuring checkpoint inhibition and anti-tumor activity during the development of cancer vaccine therapies and other biologic drugs.”
- Real-time, label-free binding analysis with surface plasmon resonance (SPR)
SPR is an optical biosensing technique that measures changes in refractive index. It involves immobilizing a ligand on the sensor surface, before introducing the analyte into the system and allowing it to pass through the flow cell. As the analyte associates with the ligand, the response signal increases, providing real-time information about the kinetics and affinity of the biomolecular interaction being investigated.
“When performing SPR experiments, one of the biggest challenges lies in optimizing the analyte concentration,” comments Campos. “To address this, we have introduced a gradient injection technology known as OneStep® into our fully automated SPR system, the Octet® SF3. This creates a gradient injection of up to four-orders of magnitude from a single analyte concentration, rather than requiring researchers to manually prepare and load multiple samples, which both saves time and reduces the risk of operator error. Advantages of SPR over established, end-point techniques like ELISA are that it allows for advanced ranking of hits using kinetic data, as well as reduces consumable use and frees up researchers’ time to be spent on other tasks.”
- Comprehensive serology with the Serum Epitope Repertoire Analysis (SERA) platform
SERA is a next-generation immunology platform that allows for examining an individual’s entire antibody epitope repertoire. It involves exposing patient sample material to a 10 billion member bacterial display peptide library before separating the antibody-binding library members for next-generation sequencing. Next, the antibody-binding peptide epitopes are compared to an epitope database containing over 26,000 human immune repertoires to identify epitopes and motifs associated with disease.
“By providing a broader, hypothesis-free view of an individual’s biology compared to assays that allow for studying only a few antigens at a time, SERA promises to reveal novel targets and aid the development of more precise clinical diagnostics,” says Shon. “Potential applications for SERA include identifying which of the malarial parasite Plasmodium falciparum’s 5,300 genes cause the most significant immune reactions, distinguishing between mild and severe cases of SARS-CoV-2, and determining the mechanisms behind immune responses to protein therapies. Additionally, SERA could be used to identify cancer via the immune response, rather than looking for circulating tumor DNA, and could improve the diagnosis of tick-borne diseases by testing for a wider range of infectious exposures than is assessed currently.”
Looking ahead
While established techniques look set to stay, researchers now have more options than ever before when deciding on an application. “From a pragmatic perspective, methods like ELISA will continue to be used for single-plex analysis,” notes Andrada. “There will always be less-funded labs and projects requiring quick answers, and these types of assays are routine.”
“The wonderful thing about research is that someone is always going to develop the next big thing, whether that be faster, cheaper, or more powerful,” says Campos. “As new technologies are developed, and researchers find increasingly innovative ways to use them, breakthrough scientific discoveries will continue being made.”