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Multiplex Immunoassays for the Detection of Extracellular and Intracellular Anyalytes Using Flow Cytometry
John M. Casper, Jennifer M. Storie, Jennifer V. Samoy, Eric B. Bertelsen and Michael C. Mullenix
Assay Designs, Inc. an Enzo Life Sciences, Inc. Company
Sponsored by Assay Designs, Inc.
Published in December 2009 (p.5) DOI:  10.2144/000113319
Abstract: Assay Designs has launched a multiplex bead immunoassay platform (MultiBead™) which incorporates flow cytometry as the assay readout. The immunoassays are carried out on the surface of fluorophore labeled polystyrene beads. The beads themselves are internally labeled with precise amounts of a single fluorophore such that separate populations of the beads can be identified by the flow cytometer based on the fluorescence intensity. Capture antibodies are coated onto the surface of the beads. Separate immunoassays can be carried out simultaneously on each different bead type. In sandwich immunoassay formats, antigen in the sample is captured by the antibody on the bead and detected using biotinylated detector antibodies. Signal is generated in the assay through subsequent addition of streptavidin labeled with a second fluorophore which emits at a different wavelength from the fluorophore in the beads. Additional multiplexing is achieved by building different immunoassays on different size beads. The flow cytometer enables simultaneous identification of bead types by size and fluorescence intensity and provides a quantitative readout of the fluorescence generated through binding of the biotinylated detector antibodies to the captured antigen. In sandwich immunoassays, as antigen concentration increases signal also increases. The platform also enables the use of competitive immunoassays formats by incorporating a labeled form of the target antigen as the detection system. In competitive assays, antigen in the sample competes with a fluorophore labeled form of the antigen for binding to the capture antibody. In competitive immunoassays the intensity of the signal is inversely proportional to the amount of antigen in the sample. The multiplex assay platform has been validated for the detection of both intracellular and extracellular human targets. The extracellular targets are proteins and small molecules involved in inflammation and include 19 cytokines and 2 eicosanoids related to inflammation. The intracellular targets are all proteins related to the heat shock response and cell signaling. The MultiBead platform also includes innovative, flexible and easy to use software for data analysis. The software is easy to customize, enables end users to assemble custom panels as well as add their own analytes. The software utilizes interpolation from point to point, 4pL or 5pL standard curve fitting to report back unknown values in a user customizable report.
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