Proteinmicroarray Formats

There are two major classes of protein microarrays: forward-phase arrays (FPA) and reverse-phase arrays (RPA) (Figure 7.2). For analysis of clinical patient specimens, we have found RPAs to be inherently superior, with many advantages over the use of FPAs.

When using FPAs, the antibodies, or bait molecules, are adherent to a substratum, such as a nitrocellulose-coated glass slide. The FPA array can, in theory, provide simultaneous information about many analytes from a single lysate, since each spot represents a different immobilized antibody. The FPA array is incubated with one patient's cellular lysate (e.g., cells procured from a tumor), and an attempt is made to measure the expression or activity of multiple proteins on one array. This approach has several obstacles that ultimately need to be overcome for optimal performance.

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FIGURE 7.2 Classes of protein-microarray technology. Forward-phase arrays (top) immobilize a bait molecule such as an antibody designed to capture specific analytes with a mixture of test sample proteins. The bound analytes are detected by a second sandwich antibody or by labeling the analyte directly (upper right). Reverse-phase arrays (bottom) immobilize the test sample analytes (e.g., lysate from laser-capture microdissected cells) on the solid phase. An analyte-specific ligand (e.g., antibody) is applied in solution phase (lower left). Bound antibodies are detected by secondary tagging and signal amplification (lower right).

FIGURE 7.2 Classes of protein-microarray technology. Forward-phase arrays (top) immobilize a bait molecule such as an antibody designed to capture specific analytes with a mixture of test sample proteins. The bound analytes are detected by a second sandwich antibody or by labeling the analyte directly (upper right). Reverse-phase arrays (bottom) immobilize the test sample analytes (e.g., lysate from laser-capture microdissected cells) on the solid phase. An analyte-specific ligand (e.g., antibody) is applied in solution phase (lower left). Bound antibodies are detected by secondary tagging and signal amplification (lower right).

Firstly, if a protein of interest is to be detected, it must be captured by the immobilized antibody, which recognizes one epitope. The array is often subsequently incubated with a second labeled antibody for detection, called a "sandwich assay." The second antibody must recognize a different epitope on the same patient's protein. The concentrations of the target protein and the antibody should be optimally matched relative to their affinity constants to identify the relevant linear detection range. Because there are two sets of affinity constants for each patient protein analyzed (corresponding to the immobilized and the second antibody), the use of this format doubles the stringency placed on detection linearity for each spot across the array. In addition, many approaches attempt to amplify the signal of the target proteins by direct conjugation with a fluorescent or biotin tag. Such conjugation often denatures, damages, or masks the epitope recognized by the immobilized antibody, and thus a blank spot on the array may not be reflective of the underlying analyte concentration.

Conversely, with the RPA (Figure 7.2), individual lysates are immobilized on the array. Each array can contain many lysates from different patient samples that are incubated with one antibody. The antibody levels are measured and directly compared across many samples. For this reason, RPAs do not require direct labeling of the patient proteins and do not utilize a two-site antibody sandwich. Hence, there is no experimental variability introduced due to labeling yield, efficiency, or epitope

Reverse Phase Protein Microarray Layout

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Experimental Samples

Phosphorylated Reference Standard

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