Multiplex assays

Song, Y, Boyer, D., Leung, L, Moheb, S., Gu, J., Doan, A.T., Khan, A., Flores, 1., Yee, F.S., and Wong, J.R., A microarray system A novel multiplexed assay platform for cytokine profiling, 95th Annual Meeting, American Association for Gancer Research, Orlando, FL, March 27-31, 2004. 

Dr. Matson currently holds seven United States patents and has contributed numerous papers in peer-reviewed journals as well as chapters in several books on microarrays. He has also made many presentations in the United States and abroad on the development of microarray technology. His current interest is in automated approaches to multiplexed assay development. 

Beads Beads are particles (made, usually, of polystyrene) that can be used as stable and inert standards for flow cytometric analysis. Beads can be obtained conjugated to various fluorochromes in order to standardize fluorescence detection settings and optical alignment or to calibrate fluorescence scales. They can also be conjugated to antibodies in order to calibrate the scale in terms of number of binding sites. More recently, in multiplexed assays, beads with capture molecules have been used to determine the concentration of soluble analytes. 

Multiplexing is challenging to the MALDI-TOF MS process. It is speculated that in multiplex assays, containing over 30 analyte molecules, a competition for available ions in the MALDI process occurs. We found that this effect can be at least partly alleviated by increasing the concentration of extend primers in the reaction. For low-plexed assays, it is recommended to use 0.5 pM of each primer whereas, for high-plexed assays, 1 pM is appropriate. 

Kuhn E, Addona T, Keshishian H, Burgess M, Mani DR, Lee RT et al (2009) Developing multiplexed assays for troponin I and interleukin-33 in plasma by peptide immunoaffinity enrichment and targeted mass spectrometry. Clin Chem 55 1108-1117... 

Keshishian H, Addona T, Burgess M, Kuhn E, Carr SA (2007) Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution. Mol Cell Proteomics 6 2212-2229... 

Wesierska-Gadek, J. et al. 2005. A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell death../. Cell. Biochem. 96, 1-7. 

Another important set of multiplexed assays monitor mRNA transcript levels. The expression level of all the genes involved in a known signal transduction pathway or other selective genes can be monitored simultaneously as a way of following compound effects on a cell. The current technologies for multiple mRNA detection include quantitative reverse transcriptional PCR (qRT-PCR), qNPA (quantitative nuclease protection assays), mass array assay technologies and branched DNA detection on Luminex beads (Panomics). The applications of such multiplexed in vitro and cell-based detection systems should provide more predicative information in hit finding and lead characterisation. 

Each microsphere is spectrally identified, and quantification is based on the intensity of the reporter signal. In comparison to conventional ELISA methods, this multiplex technology allows simultaneous quantification of up to 100 proteins in a small sample volume, and provides higher reproducibility than multiple ELISA methods. We have used fhe xMap technology to design multiplex assays for simultaneous quantification of several A and tau isoforms. Preliminary data show equal, or better, diagnostic performance than with ELISA (Lewczuk et al., submitted). 

MacAskill A, Crawford D, Graham D, Faulds K (2009) DNA sequence detection using surface-enhanced resonance Raman spectroscopy in a homogeneous multiplexed assay. Anal Chem 81 8134-8140... 

The same group has also reported a multiplexed assay based upon the Mirkin approach but in this case using an array format with non-fluorescent Raman reporters and the reporter molecule was added directly to the surface of the... 

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