Microarray antibody/immunoassay/protein

Figure 4.12 describes two popular antibody microarrays formats that are constructed for antigen capture in small sample volumes with detection by either sandwich immunoassay or antigen labeling. In sandwich immunoassay, capture antibodies are arrayed and immobilized to select specific proteins which are then found by a second labeled detection antibody. In protein target labeling, all proteins in the sample are prelabeled (i.e., fluorescent dyes) prior to capture by immobilized antibody arrays. In direct assay systems, sample proteins are directly immobilized onto... 

The most common use of protein microarrays is in immunoassays. In particular, antibody-based immunoassays are the main stream of diagnostic assays due to their specificity. The assay usually runs in a multiplexed mode where the antibodies or other capture agents are immobilized and then exposed to a biological sample. There are four immunoassay formats direct binding, sandwich (ELISA), competitive, and displacement. Direct-binding and sandwich assays are the most common. There are some reports on the use of competitive assays and displacement assays, which are usually associated with high surface area/volume systems [72-76],... 

Arenkov et al. prepared poly(acrylamide) gel pads for use in protein microarrays [199], The gels were prepared by photopolymerization of acrylamide and crosslinkers. Capture probes were immobilized, either by use of glutaraldehyde or by converting some of the acrylamide groups into hydrazides and subsequent coupling of aldehyde-modified antibodies to the pending hydrazide groups. Then, immunoassays were performed on the pads, either assays with directly labeled analytes or sandwich assays. Furthermore, the gel pads were used for enzyme activity studies. 

With the application of protein microarrays in mind, Spencer and coworkers immobilized poly(lysine) with grafted PEG side chains on various metal or semiconductor oxide surfaces via electrostatic adsorption [200], Part of the PEG side chain was functionalized with biotin at the distal end. Streptavidin was bound to the surface-tethered biotin in a subsequent step, and the remaining unoccupied binding pockets of streptavidin were then used to immobilize biotinylated capture antibodies. As an example of an immunoassay, biotinylated goat anti-rabbit IgG was immobilized, which then specifically bound rabbit IgG. 

The reverse capture autoantibody microarray is based on the dual-antibody sandwich immunoassay of enzyme-linked immunosorbent assay (ELISA) (see Fig. 1). The basic platform consists of a glass microarray slide arrayed with 1000 highly specific well-characterized monoclonal antibodies against 500 unique antigens. These antibodies are used to immobilize native proteins. Because the reagents used in the procedure are non-denaturing, antigens are... 

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