Providing Ultrasensitive Protein Quantification

Quanterix™ has created an ultrasensitive platform with the potential to measure individual proteins at concentrations 1000 times lower when compared to standard immunoassays.

The Single Molecule Array (Simoa™) technology, which is the core of this platform, allows the detection and quantification of biomarkers that were challenging or impossible to measure earlier. This paves the way for new applications to fulfill important unmet requirements in life science studies.

The Quanterix™ Homebrew Kit was developed to enable users to produce ultrasensitive assays on the Simoa™ HD-1 Analyzer in a cost-effective manner, thus improving the ability of a laboratory to deal with particular research applications of interest. The kit can be used to develop tailored digital assays most efficiently by using a lab’s own antibodies.

All the instructions and components of the kit have been meticulously validated and optimized to enhance the user-friendliness and success developing assays with the capability to achieve a sensitivity level that far exceeds that of the traditional immunoassays.

When compared to traditional enzyme-linked immunosorbent assays (ELISAs), a 2–3 log improvement in sensitivity can generally be expected. Moreover, improvement in Homebrew performance can be realized within two to three days. The Homebrew Kit was designed, keeping flexibility in mind. Since kit materials are adequate for five individual reagent batches, users can opt to either synthesize a single scaled-up batch of reagents or enhance assay sensitivity further for the target of interest.

Adequate quantities of reagents are provided for each batch for performing about 400 tests. Hence, the Homebrew Kit enables the production of more than 2,000 test results. In addition, the Homebrew Kit is provided with a Training Kit that helps confirm proficiency of preparing custom reagents, thereby enhancing productivity and success to the maximum extent.

A pre-selected antigen/antibody combination provided in the Training Kit yields expected results upon accurately performing the Homebrew Kit procedure. Lastly, the Simoa™ HD-1 Analyzer itself offers more flexibility in the development of Homebrew assays. The instrument, which is an open research platform, enables users to devise custom assay protocols and to optimize calibration curve fitting for custom assays.

The Simoa™ HD-1 Analyzer, Simoa™ Homebrew Kit, and the Homebrew Assay Development Guide together offer an exclusive and robust combination of advanced digital immunoassay technology, guided instruction, reagents, open-access tools, and complete automation to increase the productivity and efficiency to the maximum in ultra-high sensitivity immunoassay-enabled studies.

Overview of Assay Development Process

Three basic stages are involved in the Homebrew assay development process. The first stage involves the preparation of one lot of capture bead and biotinylated detector reagents. The bead and detector reagents can be considered acceptable if the bead concentrate fulfills the monomeric criterion (that is, more than 75% of beads are monomeric) and the calibration curve derived from the initial run indicates that the preferred sensitivity level has been realized.

In the second stage, additional batches may be generated for a larger quantity if the initial batch satisfies the performance targets. In the third stage, if additional sensitivity is preferred, new batches can be prepared and further optimization implemented until satisfactory performance is achieved.

Further optimization may involve the validation of the antibody’s additional conjugation levels for bead coupling, titration of assay reagent concentrations (detector antibody and streptavidin-β-galactosidase (SβG) conjugate), or the detector antibody’s additional biotinylation levels to increase the signal and minimize background to achieve further improvement in performance (see Figure 1).

The simple approach to Simoa™ assay development involves scale-up if sufficient sensitivity has been achieved or additional reagent optimization for optimal performance.

Figure 1. The simple approach to Simoa™ assay development involves scale-up if sufficient sensitivity has been achieved or additional reagent optimization for optimal performance. Image credit: Quanterix\

Additional options could be to perform an antibody flip experiment to analyze the antibody’s configurations on bead, adjustment of incubation times, variations to buffer conditions, or comparison of a 2-step assay with a 3-step assay.

High-quality Simoa™ immunoassays are similar to traditional immunoassays, and the same preferable characteristics are desired at the time of the assay development process. These characteristics are a dynamic range appropriate for the intended use of the assay, high signal:background (sound dose-response slope), and low background. The common unit of Average Enzymes per Bead (AEB) used with Simoa™ allows both digital and analog signals to be quantified to realize a stable dynamic range.1

A background signal of <2% AEB is usually sought, with the optimal background level of 0.001–0.005 AEB (0.1%–0.5%). The upper range of signal that should be focused on while developing the assay is of the order of 15–18 AEB. The CCD camera starts exhibiting saturation beyond this range. About 4–5 logs of signal range is characteristic to a Simoa™ assay.

Customer Case Study: Ultrasensitive Procalcitonin Homebrew Assay

The Natural and Medical Sciences Institute (NMI) at the University of Tuebingen, Germany, has been making use of Quanterix™’s HD-1 Analyzer and Homebrew Kit to prepare ultrasensitive assays to different biomarkers of interest. Procalcitonin is a marker of various clinical conditions, specifically bacterial infection, sepsis, and systemic inflammation. Still often, this protein’s baseline level in healthy individuals is much lower than the Lower Limit of Quantification (LLoQ) of existing sandwich immunoassays.

Various configurations of detection and capture antibody pairs were tested at the NMI by using a range of coupling concentrations for immobilizing the capture antibody, and also various biotin levels to label the detection antibody. The assay was fine-tuned by optimizing the SβG conjugate concentration. In a few weeks, NMI could realize a 100-fold increase in sensitivity than traditional ELISA (see Figure 2).

Comparison of Simoa™ and conventional ELISA. Standard curves utilizing recombinant human procalcitonin.

Figure 2. Comparison of Simoa™ and conventional ELISA. Standard curves utilizing recombinant human procalcitonin. Image credit: Quanterix\


Quanterix™’s Homebrew Kit for use with completely automated HD-1 Analyzer enables researchers to turn their current ELISA into the high-sensitivity Simoa™ platform in just a few days.

The Homebrew Kit has been modeled in such a way that any laboratory can use it to rapidly prepare custom assays of interest. It can also complement the menu of singleplex and multiplex Simoa™ assays that are available at present for a range of application areas, such as neurology, oncology, signal transduction, inflammation, metabolism, and many more.


  1. Simoa Whitepaper 1.0. Scientific Principle of Simoa™ (Single Molecule Array) Technology.

About Quanterix

Quanterix is on a mission to change the way in which healthcare is provided by giving researchers the ability to closely examine the continuum from health to disease. In order to make this vision a reality, we brought together the most experienced management team, renowned scientists, industry leading investors and expert advisors, to form a collaborative ecosystem, united through the common goal of advancing the science of precision health.

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Last updated: May 12, 2020 at 1:39 PM


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