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Liposome polymerase chain reaction assay for the sub-attomolar detection of cholera toxin and botulinum neurotoxin type A

Nature Protocols volume 1, pages 2003–2011 (2006)Cite this article

Abstract

We describe an ultrasensitive immunoassay for detecting biotoxins that uses a liposome with encapsulated DNA reporters, and ganglioside receptors embedded in the bilayer, as the detection reagent. After immobilization of the target biotoxin by a capture antibody and co-binding of the detection reagent, the liposomes are ruptured to release the reporters, which are quantified by real-time polymerase chain reaction. The new assays for cholera and botulinum toxins are several orders of magnitude more sensitive than current detection methods. A single 96-well microtiter plate can analyze ∼20 specimens, including calibration standards and controls, with all measurements conducted in triplicate. Using pre-coated and blocked microtiter plates, and pre-prepared liposome reagents, a liposome polymerase chain reaction assay can be carried out in about 6 h.

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Figure 1: Representation of a liposome detection reagent shown in cross section.
Figure 2: Results of an LPCR assay of human urine spiked with CTBS.
Figure 3: Results of an LPCR assay of deionized water spiked with BoNT/A.

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Acknowledgements

This work was supported by Army Medical Research and Material Command grant DAMD17-02-1-0178.

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Author notes

  1. Lixin Xu

    Present address: Division of Monoclonal Antibodies, Center for Drug Evaluation and Research, US Food and Drug Administration, 29 Lincoln Drive, Bethesda, Maryland, 20892, USA

Authors and Affiliations

  1. Departments of Biophysics, 1413 Research Boulevard, Rockville, 20850, Maryland, USA

    Jeffrey T Mason, Lixin Xu & Junkun He

  2. Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Boulevard, Rockville, 20850, Maryland, USA

    Zong-mei Sheng

  3. Biomedical Laboratory Research and Development Service, Veterans Health Administration, 810 Vermont Avenue NW, Washington, 20420, DC, USA

    Timothy J O'Leary

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  1. Jeffrey T Mason
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Correspondence to Jeffrey T Mason.

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Mason, J., Xu, L., Sheng, Zm. et al. Liposome polymerase chain reaction assay for the sub-attomolar detection of cholera toxin and botulinum neurotoxin type A. Nat Protoc 1, 2003–2011 (2006). https://doi.org/10.1038/nprot.2006.331

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