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Detecting antigens by quantitative immuno-PCR

Nature Protocols volume 2, pages 1918–1930 (2007)Cite this article

Abstract

The quantitative immuno-PCR (qIPCR) technology combines the advantages of flexible and robust immunoassays with the exponential signal amplification power of PCR. The qIPCR allows one to detect antigens using specific antibodies labeled with double-stranded DNA. The label is used for signal generation by quantitative PCR. Because of the efficiency of nucleic acid amplification, qIPCR typically leads to a 10- to 1,000-fold increase in sensitivity compared to an analogous enzyme-amplified immunoassay. A standard protocol of a qIPCR assay to detect human interleukin 6 (IL-6) using a sandwich immunoassay combined with real-time PCR readout is described here. The protocol includes initial immobilization of the antigen, and coupling of this antigen with antibody–DNA conjugates is then carried out by (a) the stepwise assembly of biotinylated antibody, streptavidin and biotinylated DNA, (b) the use of a biotinylated antibody and an anti-biotin–DNA conjugate or (c) the employment of an anti-IL-6 antibody–DNA conjugate. Following the assembly of signal-generating immunocomplexes, real-time PCR is used to amplify and record the signal. Depending on the coupling strategy, the qIPCR assays require 4–7 h with only about 3 h hands-on-time. The use of qIPCR assays enables the detection of rare biomarkers in complex biological samples that are poorly accessible by conventional immunoassays. Therefore, qIPCR offers novel opportunities for the biomedical analysis of, for instance, neurodegenerative diseases and viral infections as well as new tools for the development of novel pharmaceuticals.

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Figure 1: Schematic representation of the three different qIPCR options, and the ELISA technique described in this protocol.
Figure 2: Flowchart of the individual steps of the qIPCR protocol.
Figure 3: Data analysis of the qIPCR, exemplified for the sequential qIPCR detection of IL-6.
Figure 4: Typical results of the three optional qIPCR detection strategies (sequential qIPCR (gray bars), modular qIPCR (green bars) and direct qIPCR (blue bars)) for human IL-6 in human serum, compared to the analogous control ELISA (black line).

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Authors and Affiliations

  1. Universität Dortmund, Fachbereich Chemie, Lehrstuhl für Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn-Strasse 6, Dortmund, 44227, Germany

    Christof M Niemeyer

  2. Chimera Biotec GmbH, Emil-Figge-Strasse 76 A, Dortmund, 44227, Germany

    Michael Adler & Ron Wacker

Authors
  1. Christof M Niemeyer
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  2. Michael Adler
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  3. Ron Wacker
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Contributions

M.A. carried out the immunoassays, analyzed the data and wrote the manuscript; R.W. analyzed the data, prepared figures and wrote the manuscript; and C.M.N. designed and coordinated the research, analyzed the data and wrote the manuscript.

Corresponding authors

Correspondence to Christof M Niemeyer or Ron Wacker.

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Competing interests

Adler and Wacker declare employment at Chimua Biotec. Niemeyer declares financial interest as a co-founder and share holder of Chimua Biotec.

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Niemeyer, C., Adler, M. & Wacker, R. Detecting antigens by quantitative immuno-PCR. Nat Protoc 2, 1918–1930 (2007). https://doi.org/10.1038/nprot.2007.267

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