High-Throughput TechnologiesAntibody arrays: an embryonic but rapidly growing technology
Section snippets
The basics of an antibody array
Typically, antibodies are robotically delivered to the surface of a solid support and left to dry. The remaining surface is then blocked before the test sample is applied. Proteins ‘captured’ by an immobilized antibody can be identified by the addition of a second, labeled antibody that recognizes a different epitope. The appearance of a typical antibody array is shown in Fig. 2b. Kodadek [6] offers a more comprehensive review into the workings of this technology.
A technology that is opening eyes
Antibody arrays were originally conceived as multi-analyte detectors [7] but it is now clear they possess considerable potential for use in a variety of applications. Diagnosis of patient disease using this technology is stimulating much interest in, for example, leukemia [8], breast cancer [9] and preliminary studies into the diagnosis of congestive heart failure (http://www.biosite.com). Predicting the susceptibility of a patient for a particular disease is another potential clinical
Antibodies: the ideal probe?
Antibodies appear to be the most appropriate receptor element in a protein detection array because they possess the specificity required to identify a target epitope. For commercial forms of this technology, recombinant antibodies are ideal because they are derived from cell lines that are essentially immortal. They can either be intact, or in fragments, such as Fabs (fragments having the antibody binding site) or the smaller, single-chain variable fragments (scFvs). The latter are used
A suitable surface
The effective operation of an antibody array depends on the surface onto which the antibodies are printed. Surfaces that retain antibodies via hydrophobic interactions include nitrocellulose 21., 22., 23., 24. and glass slides 10., 25., 26., 27., 28., 29.. Other less conventional surfaces that could act as potential substrates for this technology include gold [30], polystyrene [31] and poly(vinylidene fluoride) (PVDF) membranes [32]. High-affinity antibody–substrate binding minimizes antibody
Variations in form
In the vast majority of antibody arrays, each dot contains a single antibody. However, Wiese et al. [10] have developed a glass plate containing multiple wells, with each well containing an 8 × 8 element array of antibodies. This novel design enables high-throughput replication of samples or simultaneous analysis of multiple samples. As this array is a prototype, it remains to be seen whether it is more effective than conventional printing of all antibodies on a single, planar surface.
De Wildt
The importance of detection
Array imaging provides a simple, qualitative confirmation of antigen–antibody binding. Detection of low-abundance proteins is more complicated. Until recently, it was accepted that amplification of protein binding, analogous to PCR in DNA arrays, was not possible using antibodies. However, signal-enhancement of bound proteins on a microarray has now been achieved using the so-called rolling-circle-amplification technique [37]. At present, proteins in antibody arrays are most sensitively
The future is still bright
Unlike DNA arrays, antibody arrays are well-designed for screening molecular interactions. Current screening of antibody–antigen interactions [41] can potentially be applied to any protein in the human proteome, provided there are corresponding antibodies. Differential proteome analysis for drug target discovery also seems attainable with antibody arrays. As proteins are commonly targeted by drugs, this technology will identify potential candidates for drug development. Furthermore, proteins
Concluding remarks
The advent of antibody arrays and their undoubted growth in the future will refine and complement existing methods of antibody–protein analysis. Some forms of this technology are already more sensitive and effective than traditional ELISA methods [21]. When the ‘ideal’ antibody array is produced, it will consist of a treated glass or silica surface that is strategically printed with different monoclonal antibodies directed against the same protein. This technology is likely to comprise the core
Acknowledgements
We would like to thank Larissa Belov for her valuable advice and support in producing this review. We also thank Lisa Nguyen for her work on Fig. 1.
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