Report from a workshop on multianalyte microsphere assays

Abstract

Multiplexed assays using fluorescent microspheres is an exciting technique that has been gaining popularity among researchers, particularly those in the public health field. Part of its popularity is due to its flexibility, as both immunoassays and oligonucleotide hybridization assays can be developed on this platform. This report summarizes a workshop held by the Centers for Disease Control and Prevention that discussed issues surrounding these assays and the Luminex 100 xMAP instrument. Topics included instrumentation, assay design, sample matrix and volume, quality control, and development of commercial applications. Cytometry (Clin. Cytometry) 50:239 -242,

Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue

“…First, as noted below, animal care and harvesting of tissues were expressly designed to minimize stress. Second, serum samples were filtered and diluted because this universally increased target protein recovery, an "unmasking effect" that may relate to removal of proteins that could trigger non-specific binding to capture and detection antibodies (Earley et al, 2002). Third, tissue samples were homogenized with sonication.…”

“…However, evaluation of user-determined protein targets can be achieved by creating appropriate specific probes sets (de Jager et al, 2003) using commercially available bead coupling kits (e.g., Amine Coupling Kit (#171-406001) from BioRad). Furthermore, bead-based assays can be designed, not only using immunoassays, but also around enzyme, receptor ligand and DNA hybridization capture molecules, further extending their utility (Dunbar et al, 2003;Earley et al, 2002). Gallagher and Appenzeller (1999) suggest that the predominant approach to scientific inquiry has been reductionist.…”

“…Recent availability of commercial instruments based on bead-based immunoassay technology (Dasso et al, 2002;de Jager et al, 2003;Dunbar et al, 2003;Earley et al, 2002;Kellar and Iannone, 2002;Kellar et al, 2001;Prabhakar et al, 2002;Vignali, 2000) is likely to help remove this void. For example, the Bio-Plex™ Suspension Array system (Bio-Rad; Hercules, CA) is an easy-to-use and flexible unit capable of simultaneously analyzing up to 100 (6-150 kDa) proteins from as little as 12 μl of sample, which has been done for analyses of serum (de Jager et al, 2003).…”

Multiplex bead array assays: Performance evaluation and comparison of sensitivity to ELISA☆

“…Interference with reliable MBAA determinations have been ascribed to anti-cytokine antibodies which may cross-react with other cytokines (3), to cross-species antibodies (3,4), as well as to other interfering substances (5). While most of these observations have been anecdotal without complete demonstration of the mechanism (s) of interference, these reports have been sufficiently frequent to indicate the problem of the matrix effect does indeed exist.…”

“…These proportions define 'spectral addresses' for each bead population. As a result, up to one hundred different detection reactions can be carried out simultaneously on the various bead populations in very small sample volumes (4,11).…”