Fluorescent multiplexed assays

Bacher JW, Elanagan LA, Smalley RL, et al. Development of a fluorescent multiplex assay for detection of MSI-High tumors. Dis Markers. 2004 20 237-250. 

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. 

Fluorescent microvolume assay technology (FMAT ) is a bead-based or cell-based fluorescent technology for homogeneous ELISA-like assays. In FMAT, a laser beam is focused on the bottom of the assay well and the localised fluorescence intensity bound to beads (or cells) is detected as an area of intense fluorescence over the unbound and background fluorescence in solution. Different analytes can be detected with appropriate fluorophores and, by using different sized beads, the assay can be multiplexed to monitor multiple analytes.13... 

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... 

An important feature in the cost per assay is the capability of easy multiplexing. Multiplexed assays are conducted together in one tube (or one well of a micro plate) and combined for example, assays are combined for different SNPs to save time and reagents. With fluorescent detection, the level of multiplexing is somewhat coupled to the amount of available dyes - which is in most applications only four. For MALDI-based SNP detection, a multiplex level of up to 12 assays has already been described [60]. 

Zyomyx (Hayward, GA) has developed the Zyomyx human cytokine biochip that uses microfluidics to measure 30 different human cytokines simultaneously. Biosite (San Diego, GA) markets commercial microfluidic chip-based immunoassay for clinical applications. Multiplexed assays using fluorescent microspheres/beads in Lab-on-Ghip format manufactured by Becton-Dickinson (Franklin, NJ), Diasorin (Stillwater, MN), and Luminex Gorp. (Austin, TX) are also available for use in clinics. 

D.A., and Imperiali, B. (2005) A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell 23. lysates. Nat, Methods, 2, 277 -284. 

Preparation of a series of phycobiliprotein tandem dyes allows multiplexed analysis of different targets in a sample. In addition, since RPE can be excited by the argon-ion laser at 488 nm, a fluorescein-labeled probe can be used concurrently with RPE alone and RPE-tandem conjugates to create a multiplexed system of different fluorescent probes that can be used simultaneously. Table 9.3 shows the different combinations of dyes that can be used in this type of assay with RPE and APC. 

The ideal assay system for HTS would include automated manipulation of single wells and each should be tested for a variety of activities simultaneously. One of the main challenges is to develop a simple assay sensitive enough to pick up enzyme activities that might be below their optimal level under the assay conditions. Nowadays novel colorimetric, luminescence, and fluorescence methods have been established in HTS with automated multiplex compound testing (typically 10-20 compounds per well) (Winson, 1997). 

Starting with the first IPCR study, gel electrophoresis retains its potential as a fast and easy method for end-point determination of DNA amplificate for IPCR assays [10, 24, 25, 29, 31, 35, 36, 38, 39, 64], Readout is performed by intercalation fluorescence markers (e.g., ethidium bromide) and photometric/densitometric quantification of band signal intensities. The direct addition of a double-strand specific intercalation marker to the PCR amplificate and subsequent measurement of fluorescence in microwells proved to be of insufficient sensitivity for the quantification of IPCR amplificate [37]. Alternative approaches, such as radioactive labeling during PCR and subsequent imaging [33], were carried out but are not well suited for routine clinical application because of additional methodological requirements. An advantage of gel electrophoresis is the possibility of simultaneous amplificate detection for multiplex IPCR [41] and the ability to detect nonspecific amplification products. 

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