Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: 10/8/2015
Publication Date: 12/8/2016
Citation: He, X., Patfield, S.A. 2016. Immuno-PCR assay for sensitive detection of proteins in real time. Methods in Molecular Biology. 1318:139-148. doi: 10.1007/978-1-4939-2742-5_14.
Interpretive Summary: This manuscript is written as a chapter for a book on immunochemical detection methods. We developed novel immuno-polymerase chain reaction (IPCR) tests for both ricin and Shiga toxins. These tests are able to detect much lower amounts of toxin than other current tests. We applied these tests to monitor concentrations of toxins in mouse blood and feces after poisoning by injection or ingestion, providing proof of principle for the use of IPCR tests in the detection of toxins in animal and environmental samples and providing a framework for developing test kits for human biological samples. Although ricin was used as sample analyte for didactic purposes in this chapter, the methodology is general and can be applied to other protein analytes important in agriculture, food safety, and health.
Technical Abstract: The immuno-PCR (IPCR) assay combines the versatility and robustness of immunoassays with the exponential signal amplification power of the polymerase chain reaction (PCR). Typically, IPCR allows a 10–1,000-fold increase in sensitivity over the analogous enzyme-linked immunosorbent assay (ELISA). This is achieved by replacing the signal-producing antibody–enzyme conjugate of an ELISA with an antibody–DNA conjugate that serves as a marker for PCR amplification. The amplification power of the PCR allows for the detection of even single molecules of nucleic acid templates, making it well suited for a broad range of applications. Here, we describe the application of an IPCR assay for detection of trace amount of antigens using ricin as an example.