The Essential Ingredients of a Modern Multiplex Immunoassay

The Essential Ingredients of a Modern Multiplex Immunoassay

The age-old adage in business is: good, fast, and cheap—pick two. For the translational scientist designing a liquid biopsy biomarker study, a viable immunoassay has the sensitivity and dynamic range to reliably detect l-protein markers from blood.. The “cheapest” assay is one that provides the most informative data for the lowest cost. Multiplexing platforms enable a cost-effective and sample-preserving method to test for multiple proteins. Likewise, “fast” and “cheap” should also consider the impact of automation on their time and other laboratory resources.

Not long ago, the biggest concern for scientists working with immunoassays was overexposure to radioactive isotopes. Today, the radioimmunoassay has largely been replaced by fluorescent-based signal readouts, including fluorophores, quantitative polymerase chain reaction (qPCR), and next-generation sequencing (NGS).

Now, researchers can focus on the real anxiety-developing questions: how many proteins do I want to measure, and will the assay miss important markers? Currently, researchers are trading off multiplex coverage for sensitivity depending on their research needs, but new technologies have emerged that are eliminating this need to “pick two.” Below, we’re discussing the essential ingredients of a modern multiplex immunoassay, specifically outlining speed and cost.

What Is an Immunoassay?

Evolved from the ELISA sandwich immunoassay, almost all methods involve “capture” antibodies (or an aptamer in the SomaScan method) that bind a target protein analyte with high affinity (see Figure 1A above). (Figure 1C). Detection of the immunocomplex depends on the binding of the second antibody and ensures specificity for and enables the concentration of the target analyte.

In single-molecule methods, which include single-molecule array (SIMOA) and single-molecule counting (SMC), detection is enabled by fluorophores conjugated to the second antibody (Figure 2A). Proximity assays, including proximity ligation assay (PLA), proximity extension assay (PEA), and nucleic acid-linked immune-sandwich assay (NULISA), rely on the hybridization of matched-pair DNA oligos conjugated to both antibodies (Figure 2B). PCR of the hybridized DNA generates the signal proportional to the protein analyte concentration. NULISA employs a biotin tag that binds to streptavidin-linked beads in a recapture immunocomplex purification step to further suppress the background signal (Figure 1D).

Components of a “Good” Immunoassay

Reliably measuring proteins in blood requires precise accounting of various markers at wildly different concentrations, called the dynamic range. Consider the blood proteome to be a bowl of Pad Thai, and your job is to account for all the food components, from the abundant noodles to the trace metals, such as the cadmium in the soil the vegetables were grown in. The challenge is simultaneously measuring the most abundant proteins that can wash out the signal of the least abundant markers that are also important.

The blood proteome encompasses a vast dynamic range, covering 12 logs of concentrations, from sub-fg/ml (attomolar) of certain cytokines to ~40 mg/ml of albumin (Melani 2022). Ideally, a multiplex assay would cover all 12 orders of magnitude without manual dilution. Commercial assays are usually either tailored to the four to five logs surrounding a protein analyte’s typical blood concentration (e.g., SIMOA and SMC) or require a manual dilution step for the most abundant proteins (e.g., PEA). NULISA demonstrates 10-12 logs of dynamic range within a single sample without dilution with their NULISAseq Inflammation Panel 250.

Assay sensitivity depends on how reliably the method can detect the least abundant proteins in the blood. Blood cells express as much as 2/3 of the human proteome, secreted proteins constitute an additional ~2000 predicted targets, while other less abundant proteins can appear in blood upon specific tissue injury. In fact, a study employing mass spectrometry, antibody-based assays, and PEA detected most of the secreted proteins in blood at low concentrations (pg/ml), while ~10 percent of predicted secreted proteins were undetected (Digre 2021).

Given that blood is the bodily fluid that bathes all the organs, assay sensitivity is crucial to providing information on the entire blood proteome, including tissue-specific markers of cell injury and early disease pathophysiology. Most assays reliably report in the pg/ml (femtomolar) range, whereas NULISA, SIMOA, and SMC can measure fg/ml concentrations for certain targets.

Speeding Up Immunoassays With Multiplexing

The speed of an immunoassay is how long it takes to measure one analyte in a given sample. Therefore, you can increase the assay speed by increasing the number of analytes in each well of a 96-well plate. Similarly, you can pool multiple specimens in a single well—bood drawn from different individuals or time points from the same donor. Confusingly, including multiple proteins and multiple samples in a single well are both referred to as multiplexing, but not all methods can account for both types of multiplexing.

Multiplex immunoassays commonly interrogate tens, or even thousands, of different proteins at once, significantly reducing the time and specimen volume required to measure the blood proteome. The single-molecule SIMOA and SMC methods employ fluorophores, which limits how many analytes you can detect at once. DNA barcode-based methods like PLA and PEA use qPCR to amplify sequences unique to each antibody, enabling hundreds or thousands of simultaneous protein measurements.

You can further exploit DNA barcodes to provide unique fingerprints for each individual sample, enabling the deconvolution of multiple specimens by next-generation sequencing. This form of multiplexing cuts down on turnaround time by reducing the number of wells that require imaging. Combining both forms of multiplexing drastically reduces the time per analyte throughout a longitudinal or population health study.

Cutting Costs With Automation

The sticker price—associated instrumentation, reagents, and kits—is the first factor to consider in assay cost. However, the most expensive attributes are often beyond the price tag. Highly skilled and trained laboratory personnel operate with multiplex immunoassays, and they critically contribute to assay costs in two ways. First, they receive a salary plus benefits for the time they spend training, designing, and running said assays. Second, they incur an opportunity cost of doing routine lab work versus something more impactful and innovative.

In biotech, the “lost science time” is often cited as a primary reason for adopting automation capabilities, and multiplex immunoassays are no exception. Most methods employ beads (e.g., magnetic) that enable immobilization to capture antibodies and subsequent automation of reagent additions, sample mixing, and washing steps. Providers have extensively aligned kits with existing liquid-handling robotics (e.g., PLA and SMC) or developed automation solutions specific to their assays (e.g., SIMOA and PEA). Depending on the options, the resulting assays require reduced hands-on time and semi- or fully-automated systems for identifying biomarkers in blood.

Another reason for automation adoption in biotech is the underlying costs associated with inter-operator and inter-sample variability. Generally, the less labor-intensive and hands-free an immunoassay is, the more you can minimize such variability. You’ll recoup untold costs when study measurements do not require repeating due to operator error, and theoretically, you’ll need fewer trial participants when minimizing inter-operator and inter-sample variability error.

Finally, there is the untold cost of missing essential biomarkers because the assay isn’t sensitive enough or doesn’t multiplex enough markers. As discussed earlier, the least abundant proteins in blood are more likely to be secreted or tissue-specific. Minute changes in their concentration may correlate with early disease onset and progression, making them invaluable biomarkers in therapy discovery and development.

The New Kid on the Block: NULISA

Choosing the right immunoassay for a specific application is often a compromise between competing biophysical limitations. For example, achieving high sensitivity comes at the cost of low multiplexing and limited dynamic range. Fortunately for translational scientists, Alamar Biosciences is addressing such limitations of the past with its groundbreaking NULISA technology. Translational scientists can now achieve a high degree of analyte and specimen multiplexing with best-in-class attomolar sensitivity over 10 logs of dynamic range (Feng 2023). Furthermore, the ARGO™ HT system offers a complete, hands-free specimen-to-data automation solution, reducing variability and, most importantly, liberating researchers to focus on the science and deliver on behalf of patients.

With a closer look at the essential ingredients of modern multiplex immunoassays, we hope you feel informed and prepared to choose Alamar as your trusted bio-tech partner.

The Essential Ingredients of a Modern Multiplex Immunoassay


Melani, et al. Science. (2022) The Blood Proteoform Atlas: A reference map of proteoforms in human hematopoietic cells. 375: 411–418.

Digre & Lindskog. Protein Science. (2021) The Human Protein Atlas—Spatial localization of the human proteome in health and disease. 30: 218–233.

Feng et al. Nature Communications. (2023) NULISA: a proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing. 14: 7238.

By: Geoffrey Feld, Ph.D., Geocyte LLC