2.9 Microsphere-Based Multiplex Immunoassays: Development and Applications Using Luminex® xMAP® Technology
The use of antibody-coated microspheres and cell sorting has been pioneered by Luminex and this is now an important immunoassay method in its own right, with many applications.
This chapter describes the Luminex range of bead-based multiplex assays, which are based on flow cytometry. These are widely used by third party manufacturers and individual researchers to develop multiplexed immunoassays for up to 500 different analytes simultaneously. The equipment and reagents available are described and the theory explained. This chapter includes detailed information to enable assays to be developed using the core system, including buffer formulations and their applications. Step-by-step protocols are provided on coupling of proteins to microspheres, confirmation of antibody coupling, and the final immunoassay (immunometric, indirect, competitive and combined immunometric/competitive). Notes are also provided on assay optimization. A list of commercially available products from third-party manufacturers is included.
2015 Update by Sherry Dunbar
Since the publication of this chapter, Luminex® has developed several tools to make the development and use of immunoassays on the xMAP® Technology platform easier than ever.
For researchers who develop or build immunoassays on a smaller scale and find that sourcing the coupling reagents can be a hassle, Luminex has created the xMAP Antibody Coupling Kit. This product contains all of the reagents and consumables needed to couple as few as 2.5 million microspheres or many as 50 million microspheres—that’s enough for 10 to 208 plates worth of samples, respectively. The kit also includes an easy to follow coupling protocol, specifically optimized for antibodies and similar proteins. More information about the xMAP Antibody Coupling Kit can be found on Luminex’s website. (www.luminexcorp.com)
For assay developers who wish to explore the possibilities of using xMAP Technology, or for those already familiar but interested in learning more about various assay designs and applications of the technology, Luminex has developed the xMAP Cookbook. This document is a collection of methods and protocols for developing a variety of assays on Luminex’s xMAP Technology. Serving as an assay development manual, whether for proteomic or genomic applications, the xMAP Cookbook is intended to help assay developers get the most out of the platform. The xMAP Cookbook can be downloaded free-of-charge from Luminex’s website at www.luminexcorp.com/cookbook.
Finally, for those who enjoy the convenience of using pre-optimized assay kits, Luminex has developed the xMAP Kit Finder. Kit Finder is an online interactive catalog of commercially available assay kits on the xMAP platform. These kits have been developed and are sold by Luminex’s partners; companies like Bio-Rad® Laboratories, EMD/Merck Millipore®, Life Technologies®, and many others. Regardless of the vendor, Kit Finder is designed to provide users of xMAP Technology with a one-stop-shop for researching and identifying the best kits to suit their testing needs. Currently, the xMAP Kit Finder is limited to immunoassays for research use only, but Luminex plans to expand the database to include all commercially available xMAP assay kits, including nucleic acid assays and diagnostic assays. xMAP Kit Finder is regularly updated and can be accessed by anyone at www.luminexcorp.com/kitfinder.
Dr. Dunbar received a Bachelor of Science degree in Microbiology and Immunology from the University of Maryland in 1986 and a Ph.D. in Basic Medical Sciences, Medical Microbiology and Immunology from the University of South Alabama, College of Medicine in 1995. Following a post-doctoral fellowship in Clinical and Public Health Microbiology at Baylor College of Medicine, she was the Laboratory Director of Virus Reference Laboratory in Houston, Texas. Dr. Dunbar joined Luminex Corporation in Austin, Texas as a Senior Scientist in 1999 and has held various scientific positions, including Senior Director of Biology Research and Development for eight years. Dr. Dunbar has developed over 100 multiplexed assay panels for detection and measurement of proteins, nucleic acids and other biomolecules on the xMAP® Technology platform. She currently serves as the Director of Scientific Affairs for Luminex.
Ms. Hoffmeyer graduated summa cum laude with a Bachelor of Science degree in Biological Sciences from Louisiana State University in Shreveport in 1999. She received her Master’s degree in Cellular and Molecular Biology from the University of Texas at Austin in 2005, where she studied inflammatory breast cancer under an NSF funded IGERT fellowship and DoD predoctoral training grant. After graduation, Ms. Hoffmeyer joined Luminex Corporation as an Applications Specialist and has held various scientific positions during her 7 years tenure. Ms. Hoffmeyer has developed numerous multiplex assays and has published and patented on several topics concerning the utility of the xMAP technology. She has brought several assay products to market, both regulated (FDA and USDA), as well as RUO. Ms. Hoffmeyer currently serves as Research and Development Manager for the Biodefense and Food Safety group at Luminex, where she directs assay development on several defense-related and integrated solution projects.
Multiplex, microsphere, bead, flow cytometry, xMAP, MagPlex, FLEXMAP 3D, Luminex, fluorescence, laser, microsphere coupling, avidin, biotin, assay development, assay optimization, immunometric, indirect, competitive.