Enzyme-linked immunosorbent assays (ELISAs) remain an extremely useful method for measuring proteins more than 50 years after their development. [1]
Relying on the amazing binding specificities of antibodies, we use ELISAs to detect proteins, hormones, and other molecules for many applications including microbial tests, cytokine measurements, and other diagnostic tests.
This article gives you a brief explanation of digital ELISAs—a different kind of ELISA assay that lets you detect several biomarkers simultaneously, at extremely low limits of detection from small sample volumes.
What is an ELISA?
ELISAs detect and quantify substances such as proteins, hormones, pathogens, and antibodies.
They rely on the specificity of antibodies to bind to the target molecule and an enzyme to make a color change, allowing for detection and measurement.
Usually, a standard curve and positive and negative controls are included in ELISA kits. This article explains ELISAs in more detail, so definitely check it out if you need to refresh yourself on them.
Introducing Digital ELISAs
Digital ELISAs represent a significant advancement in ELISA methodology. Recently, the limit of detection has been greatly lowered with the development of ultrasensitive digital ELISAs. This new kind of ELISA can detect proteins at femtomolar (10-5) levels and includes Single-Molecule Array (Simoa) technology, the Ella system, and Electrochemiluminescence ELISAs.
Simoa, similar to some flow cytometric bead arrays, is a magnetic-bead-based assay. But what makes it ultrasensitive is that the beads are spun out on a disk and settle in individual microwells, allowing enzyme-dependent fluorescence per bead to be detected.
Advantages of Digital ELISAs
Standard ELISAs are easy to perform and require no specialized equipment. However, they can only measure one analyte at a time and need larger sample volumes to run replicate wells.
Flow cytometric bead arrays can measure multiple analytes (up to 100) and require only small volumes, but they are less sensitive than digital ELISAs. Digital ELISAs, while requiring specialized equipment and being more costly, are the most sensitive and can measure multiple analytes (up to 6) with small sample volumes.
Digital ELISAs offer numerous advantages, including the ability to detect very low concentrations (up to 1000x more sensitive than traditional ELISAs) of biomarkers and save time via multiplexing.
Real-world Studies Using Digital ELISAs
Arguably, the sensitivity of detection with Simoa technology has revolutionized the field of brain injury markers the most as it has allowed for NfL, UCH-L1, Tau (including different isoforms) and GFAP to be measured in blood in traumatic brain injury as well as infections such as COVID-19 and autoimmune diseases.
These markers have great potential as readouts of therapeutic efficacy in clinical trials, for diseases such as encephalitis, Alzheimer’s, and multiple sclerosis. [2]
A comparison of three methods found that Simoa was the best for detecting the brain injury marker NfL as it was the most sensitive. [3] However, ELISAs and Simoa were found to be comparable when measuring amyloid beta peptides in the context of cerebral amyloidosis. [4]
The complexity of data analysis also increases with digital ELISAs due to the need for company-specific equipment and software. Table 1 summarizes the strengths and limitations of each method.
Table 1: Comparison of ELISAs, digital ELISAs, and flow cytometric bead arrays.
Method | ELISA | Flow cytometric Bead Array | Digital ELISA |
Strengths | Easy to perform (no specialized equipment used) | Many analytes can be measured
| Measures multiple analytes (up to 6)
|
Limitations | Only one analyte at a time
| Less sensitive than digital ELISAs | Requires specialized equipment
|
What Are Digital ELISAs Used For?
Being able to detect femtomolar levels of proteins has many advantages.
For example, when detecting brain injury markers in blood, they are usually present in very low quantities, but still significantly higher in disease conditions than healthy controls.
As a postdoc, I first started using Simoa for detecting brain injury markers in serum from research participants who had COVID-19. We found raised NfL-L in participants who had experienced a neurological complication following COVID-19, even months after infection.
So far, this technology has been used in scientific research on:
- Immunology
- Oncology
- Cardiology
- Neurodegenerative diseases
Plus, digital ELISAs have implications for many conditions, including multiple sclerosis (brain injury markers being used as a measurement of disease state) and prostate cancer (early detection of PSA).
What You Need to Get Started Using Digital ELISAs
Several different companies have their own version of the ultra-sensitive multiplex technology, each requiring its own equipment and software.
The essential lab equipment that you will need includes a temperature-controlled plate shaker, a magnetic plate washer, and multi-channel pipettes.
If you are new to digital ELISAs, you will need to get this equipment and receive instrument-specific training to be able to maintain the equipment, analyze the data, and troubleshoot issues.
What Does the Future Hold? How Low Can Detection Limits Go?
The future of ELISA technology looks promising with the continuous development of more sensitive assays with clinical implications for diagnosis and prognosis.
This is an area of active research with dropcast Simoa already having 25x greater sensitivity than Simoa as it allows even more target molecules to be measured. [5] Watch this space for the next generation of ELISA technology!
Digital ELISAs Summarized
Digital ELISAs provide higher sensitivity than regular ELISAs, allowing for the detection of proteins present in very low quantities, which is essential for early diagnosis and ongoing monitoring of a huge number of diseases.
If you’re interested in scaling up your assay to measure even more biomarkers at the same time, this article covers the related and very versatile assay: cytometric bead array.
Best of luck with pipetting and running those samples!
References
1. Engvall E, Perlmann P. (1971). Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry. 8(9):871–4
2. Wilson D, Chan D, Chang L, Mathis R, Verberk I, Montalban X, Kuhle J et al. (2024). Development and multi-center validation of a fully automated digital immunoassay for neurofilament light chain: toward a clinical blood test for neuronal injury. Clinical Chemistry and Laboratory Medicine (CCLM). 62(2):322-331
3. Kuhle J, Barro C, Andreasson U, Derfuss T, Lindberg R, Sandelius Å, Zetterberg H et al. (2016). Comparison of three analytical platforms for quantification of the neurofilament light chain in blood samples: ELISA, electrochemiluminescence immunoassay and Simoa. Clinical Chemistry and Laboratory Medicine (CCLM), 54(10):1655–61
4. De Meyer S, Schaeverbeke JM, Verberk IM, Gille B, De Schaepdryver M, Luckett ES, Poesen K et al. (2020). Comparison of ELISA-and SIMOA-based quantification of plasma Aβ ratios for early detection of cerebral amyloidosis. Alzheimer’s Research and Therapy. 12:1–16
5. Wu C, Garden PM, Walt DR. (2020). Ultrasensitive detection of attomolar protein concentrations by dropcast single molecule assays. Journal of the American Chemical Society, 142(28)
