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Advancements in protein biomarker detection technologies, such as digital enzyme-linked immunosorbent assay (ELISA), are pushing the boundaries of neurology research and diagnostics. Here, you will learn what digital ELISA is, how it works, and how researchers are using it to improve healthcare for patients with neurological disorders.

Digital ELISA vs conventional ELISA

Conventional ELISA is a common type of quantitative immunoassay technology used to measure proteins. This type of immunoassay uses ELISA microplates to immobilize a target antigen or antibody, bind with a targeted analyte, and measure an emitted signal. However, the sensitivity of conventional ELISA is often insufficient for detecting some biomarkers, especially those important in diseases such as neurological disorders and cancer. Digital ELISA is a powerful advancement in ultrasensitive immunoassays1. Digital ELISA uses beads to induce enzyme reactions in femtoliter-sized wells that can isolate and detect single enzyme molecules, allowing for orders of magnitude more sensitivity than standard sandwich-based immunoassay techniques. With digital ELISA, researchers can detect proteins at ultra-low concentrations — in the femtomolar range (fM; 10-15M) compared with nanomolar (nM; 10-9M) to picomolar (pM, 10-12M) levels of detection in conventional ELISA. Additionally, digital ELISA allows researchers to quantify proteins with minimal sample due to the low reaction volumes (15 fL well volumes in digital ELISA vs. 100 μL well volumes in conventional ELISA).

Digital ELISA empowers scientists to detect low concentration brain proteins in a variety of sample types

These enhancements in sensitivity, accuracy, and sample requirements offer a new level of flexibility and opportunity to molecular biologists, diagnostics researchers, clinical researchers, and beyond. They are particularly advantageous in neurology where they facilitate the measurement of brain biomarkers that were previously below the limit of detection and quantification with conventional immunoassay techniques. Digital ELISA helps circumvent the need for invasive sampling of cerebrospinal fluid to detect various target proteins while offering the potential to detect those same biomarkers in the blood or other non-invasively collected biofluid.

Digital ELISA Revolutionizes Neurobiology Research

Brain Biomarkers for Alzheimer’s Disease

As the most common cause of dementia, we need a faster, earlier, less invasive, and less costly method of diagnosing Alzheimer’s Disease (AD). Detection of brain biomarkers in the blood is a promising path forward. Researchers used the Simoa® platform and digital ELISA to explore the potential of blood amyloid and tau biomarkers to produce results in accordance with CSF-derived profiles in patients known to be in various stages of AD2. These researchers found that data from blood phosphorylated tau (p-tau 181) and beta-amyloid (Aβ42/Aβ40) strongly correlated with CSF-derived biomarker profiles, markers traditionally used in classifying the stage of Alzheimer’s. This research reflects the potential of blood biomarkers to be a valuable first step in accurate diagnosis for AD. One important ramification of these results is that they support the use of simple, noninvasive, and cost-effective blood-based biomarker analysis for AD research and as a diagnostic aid.

Brain Biomarkers for ALS

Amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s Disease) is a degenerative neurological disease. Diagnosis has typically relied on a complex assessment of physical traits such as walking, talking, and limb strength. Identification of specific and reliable biomarkers for ALS would improve early diagnosis, treatment and overall patient care. Falzone et al. leveraged Simoa®’s digital ELISA technology to address this unmet need3. The researchers evaluated four potential biomarkers that could be indicative of various stages or manifestations of ALS and help differentiate ALS from other neurodegenerative diseases. They evaluated GFAP (glial fibrillary acidic protein), UCHL-1 (ubiquitin carboxy-terminal hydrolase isozyme L-1), NfL (neurofilament light chain), and total tau levels in the blood. The researchers found that all biomarkers could be detected reliably in serum, at levels down to double- or even single-digit pg/mL. While UCHL-1, NfL, GFAP all proved promising biomarker candidates, NfL and UCHL1 demonstrated strong diagnostic and prognostic value in ALS patients.

Brain Biomarkers for Traumatic Brain Injury

Traumatic brain injuries can result from workplace or sports injuries, vehicle collisions, and other blunt force trauma to the head. Conventional assessments via various scans are costly, time-consuming, and, sometimes, inconclusive. A simple, rapid, and accurate blood test would allow treatment to begin sooner. Researchers found that serum biomarkers have incremental prognostic value for functional outcome after traumatic brain injury4. They examined six biomarkers (S100 calcium-binding protein B [S100B], neuron-specific enolase [NSE], GFAP, UCH-L1, NfL, and total tau) against demographic, clinical, and radiological characteristics, and over established prognostic models, such as IMPACT and CRASH. These findings support integration of serum biomarkers—particularly UCH-L1—in established prognostic models.

The Future of Brain Biomarkers with Digital ELISA

The examples above are just a few of many that have employed Simoa® digital ELISA technology to detect and accurately quantify brain biomarkers from various biofluids. The low limits of detection afforded by this technique allow researchers and clinicians to measure biomarkers that exist in peripheral biospecimens and biofluids at concentrations previously too low to be reliable.

Digital ELISA with Simoa® Technology

Quanterix offers Simoa® technology so that you can harness the power and potential of digital ELISA. Simoa® is designed for ease, speed, and ultra-low detection.

Trapping Single Molecules — Single-Molecule Detection

Simoa® is based upon the isolation of paramagnetic beads and detection of even single immunocomplexes attached to these beads, using standard ELISA reagents. The main difference between Simoa® and conventional immunoassays lies in the ability to trap single beads in femtoliter-sized wells and concentrate fluorescent signal into a small volume, allowing for a “digital” readout of individual beads to determine if they are bound to the target analyte or not.

Each molecule generates a signal that can be counted.

Creating discrete, Strong Signals

Simoa® beaded arrays use high-resolution fluorescence imaging to determine both the fraction of beads associated with at least one enzyme and the fluorescence intensity from each well. The Simoa® unit of measurement is the average number of enzymes per bead (AEB). It is calculated using either a Poisson distribution at ultra-low analyte concentrations (digital readout mode) or the average fluorescence intensity at higher concentrations (analog readout mode). The system covers all wells with an oil overlay so that diffusion is defeated to display a discrete, strong signal.

Limits of Detection — How Low Can You Go?

With its digital counting algorithm, coupled with a powerful imaging technology, Simoa® pushes the LOD and LLOQ down into the low femtomolar range.

Detect One or Many Targets with Singleplex and Multiplex Assays

With Simoa®, you can detect single (singleplex) or multiple (multiplex) targets in a single assay on a high-performance, scalable platform. By employing beads labeled with fluorophores, you can seek out up to four biomarkers at a time to study complex interactions in clinical samples. Biology is complicated. Disease is complicated. Digital Immunoassay helps you look at a problem from multiple angles at once to unravel its complexities.

Are You Ready to Detect Neurology Protein Biomarkers in Blood?

Simoa® assays can detect neurological biomarkers, such as NfL, tau, GFAP and several others associated with brain injury and disease. With Simoa®, researchers can detect these informative markers in serum or plasma, enabling earlier diagnosis and better understanding of disease pathology without invasive measures.

>Learn more about our digital ELISA technology for brain biomarkers<

References

1. Rissin DM, Kan CW, Campbell TG, et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat Biotechnol. 2010;28(6):595-599. doi:10.1038/nbt.1641

1. Delaby C, Alcolea D, Hirtz C, et al. Blood amyloid and tau biomarkers as predictors of cerebrospinal fluid profiles. J Neural Transm (Vienna). 2022;129(2):231-237. doi:10.1007/s00702-022-02474-9

1. Falzone YM, Domi T, Mandelli A, et al. Integrated evaluation of a panel of neurochemical biomarkers to optimize diagnosis and prognosis in amyotrophic lateral sclerosis. Eur J Neurol. 2022;29(7):1930-1939. doi:10.1111/ene.15321

1. Helmrich IRAR, Czeiter E, Amrein K, et al. Incremental prognostic value of acute serum biomarkers for functional outcome after traumatic brain injury (CENTER-TBI): an observational cohort study. Lancet Neurol. 2022;21(9):792-802. doi:10.1016/S1474-4422(22)00218-6