HomeSingle Molecule Counting (SMC®) TechnologyUltrasensitive Immunoassay Technology Overview

Ultrasensitive Immunoassay Technology Overview

Curious about how ultrasensitive immunoassay technology can enhance your research? Learn how Single Molecule Counting (SMC®) technology is advancing research in immunology, cardiotoxicity, neuroscience, and more. Also, explore the advantages of SMC® technology and see how researchers are using these high sensitivity assays to advance their discoveries.

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What Is SMC® Technology?

Single Molecule Counting (SMC®) technology is an advanced ultrasensitive immunoassay technology that gives researchers the power to detect biomarkers that were previously undetectable down to the femtogram/mL. It provides an indispensable tool in the researcher’s arsenal to help them move novel biology forward, fueling the discovery and development of new therapeutics. SMC® technology, originally developed by Singulex®, Inc. in 2004, relies on a basic sandwich immunoassay format utilizing two antibodies specific to the analyte of interest: a capture antibody coated on a plate or magnetic bead and a detection antibody conjugated to a fluorescent protein. Only SMC® immunoassay technology allows researchers to use both plate-based and bead-based assay designs, thus offering unmatched flexibility in assay design.

SMC® Technology Workflow

Scientists familiar with traditional immunoassays, such as ELISAs, know that sensitivity, matrix effects, and dynamic range affect how they make measurements — or if they can make them at all. Combining a traditional immunoassay workflow with patented SMC® technology enables the detection of low-abundant biomarkers, such as proteins and nucleic acids, with unparalleled sensitivity and accuracy, capturing concentrations down to the femtogram/mL level. Researchers can detect, and monitor changes in, extremely low levels of established disease biomarkers such as cardiac troponin I and cytokines.

The SMC® technology workflow has 5 steps described below. Distinguishing SMC® assays from traditional immunoassays, an elution buffer is used to break apart the immunoassay complex once constructed. The eluate containing the fluorescent reporter molecule is transferred to a 384-well plate, thus removing other assay components that contribute to high background fluorescent signal, such as the magnetic beads and antibodies used for analyte capture. The plate is loaded into the SMCxPRO® instrument, the second-generation SMC® instrument, where a laser excites the fluorescent-labeled detection antibody as it passes through a narrow interrogation window. Individual photons are captured by an avalanche photodiode and the signal is recorded. This allows for the digital quantification of individual molecules. Analyte concentrations in the unknown samples are calculated using the corresponding standard curve.

The steps in the SMC® Technology Workflow include:

Analyte of interest in solution

Sample Prep

Analyte of interest in solution

    Capture on plate or bead

    Capture on plate or bead

      Antibodies translate each biomarker into a signal

      Antibodies translate each biomarker into a signal


        Complex is chemically broken apart


          Sample is detected by laser and detection tags are counted 

            After following a traditional sandwich ELISA workflow, the proprietary SMC® protocol steps concentrate the signal by disassociating the fluorescent-labeled detection antibody from the sandwich complex. The fluorescent-labeled detection antibody is the signal acquired in the SMCxPRO® instrument. This results in reproducible signal, and improved quantification of proteins, particularly those at very low abundance. With better precision and sensitivity, researchers can:

            • Quantify previously undetectable analytes
            • Better stratify sample populations
            • Gain insights into novel biological mechanisms
            • Require fewer data points for critical decision making
            • Accelerate drug discovery and development
            • Reduce program costs and improve productivity

            Ultrasensitive Biomarker Detection Platform

            SMC® technology offers an ultrasensitive biomarker detection platform with the flexible SMCxPRO® platform. Table 1 compares running assays on this platform to running traditional ELISAs.

            Table 1.Comparison of SMC® assays on the SMCxPRO® platform to traditional ELISAs.

            Ancillary Equipment and Kits to Enhance the SMC® Workflow

            To enhance the SMCxPRO® platform, we also offer ancillary equipment and kits specifically for SMC® technology, such as:

            Research Applications

            Ultrasensitive immunoassays can enhance research in a variety of fields because the improved sensitivity allows researchers to dive deeper into their studies. Research applications of SMC® technology include the areas of:

            Frequently Cited Advantages of SMC® Technology

            Single Molecule Counting (SMC®) technology provides maximum immunoassay performance while following a workflow very similar to traditional ELISA technology.

            Traditional ELISA methodologies demonstrate limitations in sensitivity and dynamic range, typically require high sample volumes, and are susceptible to matrix effects. Combined, these factors reduce the utility of traditional ELISAs for the detection of low-abundant proteins and endogenous biomarker levels in healthy subjects, thus hampering statistical analysis among study groups. By adapting an ELISA workflow, SMC® technology achieves improved signal-to-noise ratios over traditional immunoassay technologies, thus providing quantification at both low and high levels of expression in one complete system. Digital counting of fluorescent events improves the assay sensitivity and extends the assay dynamic range beyond what can be achieved with traditional immunoassays.

            Because SMC® immunoassay technology can reach fg/mL sensitivity ranges, this platform offers the ability to dilute pre-clinical samples, when only low sample volumes are available. 

            Users are fully supported by onsite Immunoassay Field Application Scientists and Specialists, as well as dedicated technical support teams.

            We understand the SMC® platform is an important investment for research labs and is committed to ensuring the success of its users. Regardless of the types of assays being used, all SMC® users are fully supported by onsite Immunoassay Field Application Scientists and Specialists who have experience working with researchers from a broad range of lab types, including academic, government, biotech, pharma, CRO, and regulated labs.

            The SMC® platform is a versatile system that can be used in multiple study types, including biomarker assessment, bioanalytical work such as pharmacokinetic and pharmacodynamic studies, and immunogenicity testing.

            Complimenting a menu of off-the-shelf, verified assay kits, our Custom Assay Development and Innovation (CADI) team can be contracted to perform custom assay development and sample testing services at their site in St. Louis, MO, USA. Request more information about our custom services on our CADI page.

            SMC® assays are available in bead-based formats and can be developed in both plate-based and bead-based formats by our CADI team.

            The proprietary SMC® technology allows scientists to measure proteins with increased precision, enabling unparalleled quantification at low and high abundant levels of expression. The flexible SMC® immunoassay system acquires data from both plate-based assays and bead-based assays, providing a choice of format depending on budget and quantification requirements. Learn more about how we develop custom SMC® assays in our article on CADI services.

            The SMC® assay read plate is a 384-well plate and the entire plate can be read in less than three hours. This high-throughput platform allows researchers to perform an entire SMC® assay run from sample prep through data analysis in one day.

            SMCxPRO® system can be integrated with a Hamilton Microlab® STARlet liquid handing system, increasing assay throughput.

            In certain environments, automation of SMC® immunoassays is desirable so that researchers can focus on other high-value activities to increase overall efficiency. The Hamilton Microlab® STARlet liquid handling workstation offers a hands-free option providing a robust, reproducible SMC® workflow eliminating sources of error and variability. Our Custom Assay Development and Innovation (CADI) team uses this technology when applicable to reduce lead time on sample testing projects.

            Both data acquisition and analysis are performed within a single software package.

            The SMCxPRO® software package was developed in-house, thus affording full transparency to data processing algorithms. The software is user-friendly and allows end-users to set up the instrument, read the plate, and analyze results quickly and easily. The software enables easy data curation, including manual outlier removal.

            For labs operating in a regulated environment, the SMCxPRO® instrument generates one signal data stream that can be imported into Laboratory Information Management Systems, such as WATSON, or other software. 21 CFR Part 11 compliance features can also be enabled. 

            For Research Use Only. Not For Use In Diagnostic Procedures.

            Curious for more? Learn about the capabilities of our multiplex immunoassays on our MILLIPLEX® multiplex assay overview page.

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