Multiplexed protein profiling on microarrays by rolling-circle amplification

Abstract

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 detection via direct enzymatic amplification of short DNA aptamers

“…In addition, RCA amplification of aptamer-hybrids can be readily adapted for protein detection in chip-based applications as the signal is spatially localized at the site of the binding event. (33) …”

“…In addition, immuno-DNA detection strategies have been extended to use rolling circle amplification (RCA), an isothermal technique that generates a long ssDNA oligomer tethered to the immuno-DNA conjugate. (31)(32)(33)(34) A drawback of these approaches is that the synthesis of the antibody-DNA hybrids can be problematic as controlling the location and number of DNA conjugates per protein is not always straightforward, often leading to heterogeneous ratios of DNA tags per antibody. Recent developments in site-specific conjugation of oligonucleotide tags to proteins using intein chemistry (or chemical ligation) have been very successful, (35) although conjugate preparation remains laborious.…”

“…As both recognition and readout capabilities are incorporated into a single DNA molecule, tedious conjugation procedures required for protein-DNA hybrids are omitted. As for the amplification process, the DNA reagent can be directly subjected to either PCR (27,30) or RCA (31)(32)(33)(34) without the need for additional ligation or modification reactions. To demonstrate the approach, we designed a 50nt ssDNA molecule, which was comprised of an aptamer domain, a poly[A] 15 linker, and a short 20nt primer ( Figure 1B).…”

Array-based proteomics: mapping of protein circuitries for diagnostics, prognostics, and therapy guidance in cancer

“…In order to cover the wide variety of analyte concentrations in a sample, serial dilutions are necessary in a forward-phase array format. To address this and to increase the throughput of sandwiched assays, several authors have successfully used antibody arrays in microtitre plates [in essence, a variant of enzyme-linked immunosorbent assay (ELISA) [29]] and on sub-compartmentalized glass slides [30].…”

Proteomics in human cancer research

“…The second approach is a sandwich immunoassay, which is opposite to the first method. In this case, antibodies are immobilized on the solid support, and bound proteins are detected using a second labeled antibody [64]. The third method is an antigen capture assay and is similar to the second approach.…”