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Noncompetitive phage anti-immunocomplex real-time polymerase chain reaction for sensitive detection of small molecules.

Authors
Kim-HJ; McCoy-M; Gee-SJ; González-Sapienza-GG; Hammock-BD
Source
Anal Chem 2011 Jan; 83(1):246-253
NIOSHTIC No.
20038594
Abstract
Immuno polymerase chain reaction (IPCR) is an analytical technology based on the excellent affinity and specificity of antibodies combined with the powerful signal amplification of polymerase chain reaction (PCR), providing superior sensitivity to classical immunoassays. Here we present a novel type of IPCR termed phage anti-immunocomplex assay real-time PCR (PHAIA-PCR) for the detection of small molecules. Our method utilizes a phage anti-immunocomplex assay (PHAIA) technology in which a short peptide loop displayed on the surface of the M13 bacteriophage binds specifically to the antibody-analyte complex, allowing the noncompetitive detection of small analytes. The phagemid DNA encoding this peptide can be amplified by PCR, and thus, this method eliminates hapten functionalization or bioconjugation of a DNA template while providing improved sensitivity. As a proof of concept, two PHAIA-PCRs were developed for the detection of 3-phenoxybenzoic acid, a major urinary metabolite of some pyrethroid insecticides, and molinate, a herbicide implicated in fish kills. Our results demonstrate that phage DNA can be a versatile material for IPCR development, enabling universal amplification when the common element of the phagemid is targeted or specific amplification when the real time PCR probe is designed to anneal the DNA encoding the peptide. The PHAIA-PCRs proved to be 10-fold more sensitive than conventional PHAIA and significantly faster using magnetic beads for rapid separation of reactants. The assay was validated with both agricultural drain water and human urine samples, showing its robustness for rapid monitoring of human exposure or environmental contamination.
Keywords
Laboratory-techniques; Laboratory-testing; Bioassays; Immunological-tests; Exposure-assessment; Environmental-health-monitoring; Monitoring-systems; Antibody-response
CODEN
ANCHAM
Publication Date
20110101
Document Type
Journal Article
Email Address
bdhammock@ucdavis.edu
Funding Type
Agriculture; Cooperative Agreement
Fiscal Year
2011
NTIS Accession No.
NTIS Price
Identifying No.
Cooperative-Agreement-Number-U50-OH-007550
Issue of Publication
1
ISSN
0003-2700
Source Name
Analytical Chemistry
State
CA
Performing Organization
University of California - Davis
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