Guide to Bead-Based Immunoassays
Immunoassays have had a significant impact on life sciences research and clinical diagnostics.
Radioimmunoassays, Western Blots, and ELISAs have contributed to the detection and measurement of critical biomarkers. However, each of these methods has shortcomings. The ideal immunoassay is highly sensitive, can measure multiple biomarkers, and produces consistently reproducible results.
Enter Simoa® bead-based immunoassays. These assays have helped transform the study of human health and disease by facilitating disease-associated biomarker detection and better treatment response monitoring.
To fully appreciate the revolutionary contribution of this new technology, it is essential to first understand the strengths and limitations of preceding immunoassay technology.
A Brief History of Immunoassays
Radioimmunoassay (RIA), Yalow & Berson1
The first immunoassays used radioisotopes in a competitive assay to detect a single antigen of interest. Because of its remarkable sensitivity, RIA remained the standard for detection for over a decade despite the health and disposal risks.
Western Blot, Towbin, Staehlin, & Gordon2
Western blots, like RIAs, are qualitative assays that traditionally detect a single protein. They involve transferring biological samples from an electrophoresis gel to the surface of a membrane. The resulting bands present semi-quantitative data. However, the high susceptibility to kinetic differences between runs results in low reproducibility.
Enzyme-linked immunosorbent assay (ELISA), Engvall & Perlmann3, Van Weeman & Schuurs4
The advent of ELISA technology introduced the first truly quantitative approach to protein detection. Offering multiple assay structures and detection methods, ELISAs offered specificity and sensitivity that wasn’t previously attainable. However, ELISA technology is still limited by its ability to only detect and measure one analyte at a time.
| Are you curious about the differences between Simoa bead-based immunoassays and ELISAs, which one is more sensitive, and when you should use each? Learn the Difference. |
Bead-Based Immunoassays
Bead-based immunoassays build upon ELISA technology to offer precise singleplex and multiplex solutions without compromising sensitivity, accuracy, or reproducibility. Unlike ELISAs that use a plate’s surface as the site of enzyme-antigen binding, bead-based immunoassays use antibody-coated beads.
Quanterix’s single-molecule assays (Simoa®) use paramagnetic beads coupled with antibodies that bind to a specific target analyte. During the initial phase of the assay, an immunocomplex forms consisting of a bead, a bound protein, a detection antibody, and a reporter enzyme. Next, this mixture is added to a disc containing approximately 240,000 microwells perfectly sized to admit a single bead. Finally, excess beads are washed away, and an oil solution is added to seal the beads within individual wells.
Fluorescent enzymatic signal amplification reveals the presence of a bound protein. Simoa® bead-based immunoassays are particularly well suited to quantifying low-concentration samples, as each bead only needs one bound protein for the well to fluoresce. Because the volume inside the microwells is so small, fluorescence quickly accumulates and can be measured above background. In addition, unique bead sets can be encoded with a distinct fluorescent dye specific to a biomarker of interest, enabling simultaneous measurement of multiple analytes.
| Guide: Which Immunoassay Is Right for Your Research? |
|---|
| There’s a lot to consider when designing experiments. Use our guide to determine which questions you should ask when selecting an immunoassay and a detection method so you can make the right choice for your research objectives. Read the guide. |
Simoa® Technology and Your Research
Quanterix’s singleplex and multiplexed bead-based immunoassays achieve ultra-sensitivity levels through digital measurements of bound immunocomplexes. In addition, Simoa®’s ability to count single protein molecules allows researchers to measure critical biomarkers previously undetectable using other methods.
What could you do with this technology?
Quantify more biomarkers and strengthen your publication
Whether you are looking to analyze one analyte or many, Quanterix offers ready-to-use Simoa® assay kits that save you time and contain all the reagents needed to detect your target biomarkers. Simoa®’s neurology bead-based multiplex assays can measure multiple analytes simultaneously, allowing you to include analysis of multiple biomarkers in your publication without significantly increasing your bench time.
Detect biomarkers faster and increase your experimental output
Quanterix’s HD-X™ Automated Immunoassay Analyzer delivers results in an automated fashion using 96 well plates or tubes containing plasma, serum, cerebral spinal fluid (CSF), cell lysate, urine, or others, reducing sample processing and transfer time. With such an instrument, you can perform more experiments with less hands-on time and higher reproducibility. For lower throughput use and smaller footprint, the SR-X™ Biomarker Detection System enables semi-automated workflow and high flexibility for assay development.
Benefit from higher sensitivity and more effective sample usage
One of the biggest hurdles in immunoassay technology development is increasing detection and measurement sensitivity. Simoa®’s sensitivity is significantly greater than conventional immunoassays and represents a broad dynamic range allowing for the exploration of previously undetectable biomarkers.
Typical sample volumes for Simoa® bead-based immunoassays are between 50µl and 150µl, comparable to ELISA. However, sample usage becomes more efficient in a multiplex assay that detects multiple analytes in a single sample.
Enjoy a more productive workflow with high reproducibility and less troubleshooting
Most Simoa® assays go through analytical validation for plasma, serum, and CSF to assure high reproducibility so you spend more time experimenting and less time troubleshooting. What’s more, running assays with higher sensitivity enables you to measure a higher percentage of samples, including controls. This results in more robust stratification between study groups to reach significant findings that would otherwise not be possible. High sensitivity also allows higher sample dilution, which can help with sample matrix effects and limited sample volumes.
Develop custom assays tailored to your research needs
Existing ready-made assays should not define your biomarker selection. However, custom assays can be costly, frustrating, and labor intensive. This hurdle causes some researchers to accept pre-made kits and limited project scope.
To address this, we developed Simoa® Homebrew Kits so you don’t have to compromise. Like the ready-to-use assay kits, homebrew kits come with all the reagents and instructions you need to develop customized assays to be used on Simoa® platforms. We’re giving you the power to develop the assays that fit your research needs. What will you do with it?
Learn how Simoa ultrasensitive technology can revolutionize your research!
References
- YALOW RS, BERSON SA. Immunoassay of endogenous plasma insulin in man. J Clin Invest. 1960 Jul;39(7):1157-75. doi: 10.1172/JCI104130. PMID: 13846364; PMCID: PMC441860.
- Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4. doi: 10.1073/pnas.76.9.4350. PMID: 388439; PMCID: PMC411572.
- Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry. 1971 Sep;8(9):871-4. doi: 10.1016/0019-2791(71)90454-x. PMID: 5135623.
- Van Weemen BK, Schuurs AH. Immunoassay using antigen-enzyme conjugates. FEBS Lett. 1971 Jun 24;15(3):232-236. doi: 10.1016/0014-5793(71)80319-8. PMID: 11945853.