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.
Abstract
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.
Contributors
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.
Keywords
Multiplex, microsphere, bead, flow cytometry, xMAP, MagPlex, FLEXMAP 3D, Luminex, fluorescence, laser, microsphere coupling, avidin, biotin, assay development, assay optimization, immunometric, indirect, competitive.