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Multiplexed protein profiling on microarrays by rolling-circle amplification

Nature Biotechnology volume 20, pages 359–365 (2002)Cite this article

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 accommodate ∼1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys.

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Figure 1: Schematic representation of immunoassays with RCA signal amplification.
Figure 2: Sensitivity of cytokine detection by RCA and direct detection.
Figure 3: Kinetics of cytokine production in maturing LCs on microarrays.
Figure 4
Figure 5: Comparison of MDC measurement in supernatants by commercial ELISA and multiplexed RCA-amplified microarray immunoassay.

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Acknowledgements

We thank K. Kukanskis and S. Cooley (MSI) for manufacturing the protein microarrays used in this study. We also thank Ron Lennox for suggesting the collaboration between Molecular Staging, Inc. and Cellular Genomics, Inc.

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  1. Barry Schweitzer

    Present address: Protometrix, Inc., 66 High Street, Guilford, CT, 06437

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  1. Molecular Staging, Inc., New Haven, 06511, Suite 701, 300 George Street, CT

    Barry Schweitzer, Brian Grimwade, Weiping Shao, Minjuan Wang, Qin Fu, Quiping Shu, Isabelle Laroche, Zhimin Zhou, Velizar T. Tchernev, Jason Christiansen & Stephen F. Kingsmore

  2. Cellular Genomics, Inc., 36 East Industrial Road, Branford, 06405, CT

    Scott Roberts & Mark Velleca

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Correspondence to Stephen F. Kingsmore.

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Schweitzer, B., Roberts, S., Grimwade, B. et al. Multiplexed protein profiling on microarrays by rolling-circle amplification. Nat Biotechnol 20, 359–365 (2002). https://doi.org/10.1038/nbt0402-359

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