Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1β, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-α treatment. Because microarrays can accommodat ~1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys.Several recent reports have established the feasibility of protein arrays for a variety of applications [1][2][3][4][5][6][7] . To meet the emerging needs of expression proteomics, however, such arrays must yield highly multiplexed, sensitive, quantitative, and reproducible measurements of protein levels. It is also desirable that assays on these arrays utilize small sample volumes and be compatible with hardware and software used by the DNA microarray industry. Microarrays of ordered immobilized capture antibodies and attendant sandwich immunoassays are a straightforward, near-term approach for highly parallel measurement of protein levels. Polyclonal or monoclonal antibodies for several thousand proteins are available, and are being supplemented with affinity probes generated by phage and ribosomal display, affibodies, and aptamers [8][9][10][11] . Indeed, recent studies have described sensitive 12,13 RCA is a useful alternative for on-chip signal amplification [15][16][17] .It permits sensitive and highly multiplexed assays on microarrays because RCA-amplified signals remain localized at the microarray spot ( Fig. 1) 16,17 . When utilized on microarrays of printed proteins, RCA has been shown to allow detection of protein analytes with zeptomole sensitivity and broad dynamic range 16,18,19 . In the present study, we establish the utility of RCA for highthroughput analysis of protein expression on microarrays, providing assays that are highly sensitive, quantitative, and reproducible. We describe highly multiplexed, microarray immunoassays with four steps: sample application and...

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.

Background: Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification.

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