Optimization of Rolling-Circle Amplified Protein Microarrays for Multiplexed Protein Profiling

Abstract

Protein microarray-based approaches are increasingly being used in research and clinical applications to either profile the expression of proteins or screen molecular interactions. The development of high-throughput, sensitive, convenient, and cost-effective formats for detecting proteins is a necessity for the effective advancement of understanding disease processes. In this paper, we describe the generation of highly multiplexed, antibody-based, specific, and sensitive protein microarrays coupled with rolling-circle signal amplification (RCA) technology. A total of 150 cytokines were simultaneously detected in an RCA sandwich immunoassay format. Greater than half of these proteins have detection sensitivities in the pg/ml range. The validation of antibody microarray with human serum indicated that RCA-based protein microarrays are a powerful tool for high-throughput analysis of protein expression and molecular diagnostics.

Figure 1

Schematic representation of microarray immunoassay with RCA signal amplification. It consists of (a) analyte capture by antibody immobilized on microarray, (b) detection by biotinylated secondary antibody, (c) binding of antibiotin antibody-oligonucleotide conjugate pre-annealed with circle 1, and (d) rolling-circle replication to generate long single-strand DNA which is hybridized with oligonucleotide decorator.

Figure 2

Optimization of antibodies from different sources (A and B) for choosing optimal pair for immunoassay on protein microarray with examples of (a) IL-13 and (b) MCP-2.

Figure 2

Optimization of antibodies from different sources (A and B) for choosing optimal pair for immunoassay on protein microarray with examples of (a) IL-13 and (b) MCP-2.

Figure 3

Titration of SSB in the RCA reaction mixtures. Cytokine IL-8 and Eot-2 at 100 pg/mL were detected.

Figure 4

Cross-reactivity of capture antibodies and analytes ((a) and (c)), capture antibodies and detection antibodies ((b) and (d)) on the subarrays 3 (A) and 4 (B). Each subarray was printed with about 35 different capture antibodies (listed on the chart). Cross-reactivity was determined by using different matched secondary antibody and 50 ng/mL or 0 ng/mL of its cognate analyte, which has been described in the text. A shaded square off the diagonal indicates the presence of nonspecific signals (intensity ≥ 300). None of these nonspecific signals exceed the intensity of 600 fluorescence intensity units (images are scanned at PMT of 600 and power of 100% on Axon scanner).

Figure 4

Cross-reactivity of capture antibodies and analytes ((a) and (c)), capture antibodies and detection antibodies ((b) and (d)) on the subarrays 3 (A) and 4 (B). Each subarray was printed with about 35 different capture antibodies (listed on the chart). Cross-reactivity was determined by using different matched secondary antibody and 50 ng/mL or 0 ng/mL of its cognate analyte, which has been described in the text. A shaded square off the diagonal indicates the presence of nonspecific signals (intensity ≥ 300). None of these nonspecific signals exceed the intensity of 600 fluorescence intensity units (images are scanned at PMT of 600 and power of 100% on Axon scanner).

Figure 5

Protein expression profiling of normal human serum (Sigma) on 70-feature antibody microarray. Specific signals detected were consistent with the protein level in serum. (a) Images of detecting 70 proteins in antibody microarray obtained with Axon microarray scanner. (b) Fluorescence intensities were derived from microarray images with averaging four spots of the same feature.

Figure 5

Protein expression profiling of normal human serum (Sigma) on 70-feature antibody microarray. Specific signals detected were consistent with the protein level in serum. (a) Images of detecting 70 proteins in antibody microarray obtained with Axon microarray scanner. (b) Fluorescence intensities were derived from microarray images with averaging four spots of the same feature.

Figure 5

Protein expression profiling of normal human serum (Sigma) on 70-feature antibody microarray. Specific signals detected were consistent with the protein level in serum. (a) Images of detecting 70 proteins in antibody microarray obtained with Axon microarray scanner. (b) Fluorescence intensities were derived from microarray images with averaging four spots of the same feature.