Assays microarray-based

Mendoza, L.G., McQuary, R, Mongan, A., Gangadharan, R., Brignac, S., and Eggers, M., High-throughput microarray-based enzyme-linked immunosorbent assay (ELISA), Biotechiques, 24, 778-788, 1999. 

Such thermoplastics have also been used as DNA microarray substrates (Matson et al., 1995 Shchepinov, 1997 Beier and Hoheisel, 1999) and in the construction of protein microarrays in microwells (Matson et al., 2001 Moody, 2001). Pierce (see searchlight perbio.com) introduced the Search-Light series of microarray-based ELISA assays immobilizing capture antibodies in a low density array format into polystyrene microwells. 

Matson, R.S., Milton, R.G., Gress, M.D., and Rampal, J.B., Microarray-based cytokine immunosorbent assay. Oak Ridge Gonference, 2001, Poster 20. 

Two important observahons were noted. First, no single microarray substrate satisfied all performance criteria for all antibodies. Second, in other work, certain antibodies worked well in conventional ELISA but not in microarray format. Thus, results from ELISA do not necessarily qualify an antibody for use on microarrays. If is besf to validate each antibody for use on the intended substrate in a microarray-based assay. 

The issue of which antibody to select for an assay is not a new problem. Certainly anyone involved in the development of an immunoassay has been faced with this choice. Consider attempting to create a multianalyte, microarray-based micro-ELISA of modest density (10 to 100 analytes) and determining which capture antibodies to use based upon their affinities, stabilities, and cross-reactivities. For a sandwich assay, add in the 10 to 100 analyte-specific secondary (reporter) antibodies and determine their levels of cross-reactivity with each other and with the specified antigens and capture antibodies. In other words, achieving high performance for all analytes with a microarray immunoassay is indeed a formidable challenge. 

Recent detection methods for glycan array include fluorescent assay, SPR, MALDI-TOF mass spectrometry, and nanoparticle assay. Fluorescence-based measurement is the prevalent principle for detecting binding to glycan microarrays. Rhodamine [9],... 

The microarray assay is based on hybridization reactions between labeled single stranded molecules in solution (target) and complementary molecules immobilized on the flat surface of the slide (probe). The fabrication of a microarray requires the synthesis of the target and its deposition on the slide surface (deposition technology). Alternatively, a different approach involving the synthesis of the target directly onto the surface can also be employed. 

In addition to commercially production, a great deal of research and development work on biochips has been going on both in industry and in academia. Genomic analysis of DNA and RNA continues to be the focus of interest, but more and more effort is being spent on proteomic analysis of proteins and peptides. Several enzyme assays and immunoassays designed based on microarray-based systems with simple microfluidic control are close to commercialization. They can be a vital tool in clinic diagnostics, drug discovery, and biomedical research. 

Fig. 3 Typical microarray-based assay. A microarray is incubated with a solution containing multiple target molecules. After removal of unbound molecules, the target-bound microarray is stained with fluorescence, followed by imaging and quantification...

Volokhov, D., A. Rasooly, K. Chumakov, and V. Chizhikov. Identification of Listeria species by microarray-based assay. J Clin Microbiol. 2002, 40(12), 4720-4728... 

Multiplexed immunoassays, which permit the simultaneous assay of several analytes in the same sample, are already available commercially. These systems typically depend on the use of reagent beads functionalized with different capture antibodies directed against the analytes of interest. Interest in multiplexed assays seems likely to continue to develop rapidly. The advantages and limitations of bead-based and microspot/microarray-based multiplexing approaches, as well as their application to the field of molecular diagnostics, have been discussed recently [200]. 

The development of an automated selection process, the intrinsic properties of aptamers combined with their conversion into signaling elements, and the ease of conjugating oligonucleotides on different surfaces make them appropriate for their use in microarray formats. Interest in aptamer chips was outlined long ago (Brody and Gold, 2000). They could be used for the identification and quantification of multiple proteins or biomarkers. There are still few reports on the development of microarray-based aptamer assays. Detection of the analyte... 

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