For many years, ELISA has been the method of choice for a variety of applications in the research community, and for good reason. It is easy to perform and delivers great results when performed correctly. However, there are many benefits that Luminex’s bead-based immunoassay method, known as xMAP Technology, offers over the traditional ELISA.
In a video format, this article illustrates just how easy it is to take a perfectly good ELISA and make it better; by converting it over to the xMAP platform. In this experiment, Luminex scientists take a standard off-the-shelf ELISA kit and port it over to this platform; along the way, demonstrating how the multiplex capabilities of the technology can help you optimize your assay by finding antibody pairs that perform the best.
In addition to performance benefits such as improved dynamic range, sensitivity, and time to result, there is also a significant cost savings that comes with transitioning your existing ELISA to xMAP Technology. Due to more efficient use of antibody in the xMAP assay, it is not uncommon to see a near 50% reduction in cost per sample. When more than one analyte is multiplexed together, the cost and time savings grow exponentially.
Featured in the experiment are a few Luminex products that make this ELISA conversion even easier.
- MAGPIX® Instrument
MAGPIX is the latest instrument from Luminex. Capable of measuring up to 50 targets simultaneously, it is the lowest cost xMAP instrument on the market, making it ideal for those who want to incorporate multiplexed methods into their lab for the first time.
- Low Concentration MagPlex® Microspheres
Now available in a more convenient concentration (2.5 million beads/mL), MagPlex Microsphere vials contain the right amount of beads for early ELISA to xMAP conversion experiments as well as lower throughput applications.
- xMAP® Antibody Coupling Kit
This simple kit is perfect for ELISA to xMAP Technology conversion. It includes all the reagents and consumables required to quickly and easily get your antibody of interest on to microspheres; allowing you to spending less time searching for vendors and testing buffer formulations, and more time conducting your research.
- Luminex® Magnetic Tube and Plate Separators
Whether washing an entire plate of samples or a single tube, these magnetic separators are just what you need to make coupling and assay preparation fast and easy.
The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest. The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; low-concentration magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest.
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.
Cite this Article
Baker, H. N., Murphy, R., Lopez, E., Garcia, C. Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method. J. Vis. Exp. (), e4084, DOI: 10.3791/4084 (2012).