title: Detecting respiratory syncytial virus using nanoparticle-amplified immuno-PCR.
abstract: Early-stage detection is essential for effective treatment of pediatric virus infections. In traditional -immuno-PCR, a single antibody recognition event is associated with one to three DNA tags, which are subsequently amplified by PCR. In this protocol, we describe a nanoparticle-amplified immuno-PCR assay that combines antibody recognition of traditional ELISA with a 50-fold nanoparticle valence amplification step followed by amplification by traditional PCR. The assay detects a respiratory syncytial virus (RSV) surface fusion protein using a Synagis antibody bound to a 15 nm gold nanoparticle co-functionalized with thiolated DNA complementary to a hybridized 76-base Tag DNA. The Tag DNA to Synagis ratio is 50 to 1. The presence of virus particles triggers the formation of a "sandwich" complex comprised of the gold nanoparticle construct, virus, and a 1 μm antibody-functionalized magnetic particle used for extraction. Virus-containing complexes are isolated using a magnet, DNA tags released by heating to 95 °C, and detected via real-time PCR. The limit of detection of the nanoparticle-amplified immuno-PCR assay was compared to traditional ELISA and traditional RT-PCR using RSV-infected HEp-2 cell extracts. Nanoparticle-amplified immuno-PCR showed a ∼4,000-fold improvement in the limit of detection compared to ELISA and a fourfold improvement in the limit of detection compared to traditional RT-PCR. Nanoparticle-amplified immuno-PCR offers a viable platform for the development of an early-stage diagnostics requiring an exceptionally low limit of detection.
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN, USA.
authors.forename: Jonas W
authors.initials: JW
authors.lastname: Perez
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Adams
authors.forename: Grant R
authors.initials: GR
authors.lastname: Zimmerman
authors.forename: Frederick R
authors.initials: FR
authors.lastname: Haselton
authors.forename: David W
authors.initials: DW
authors.lastname: Wright
mesh_terms: D000914:Antibodies, Viral / Q000737:chemistry / Q000276:immunology; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D006046:Gold / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods*; D057230:Limit of Detection; D059346:Magnets / Q000737:chemistry; D053768:Metal Nanoparticles / Q000737:chemistry*; D008863:Microspheres; D058525:Quartz Crystal Microbalance Techniques; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012136:Respiratory Syncytial Viruses / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D000914:Antibodies, Viral; D004279:DNA, Viral; D012367:RNA, Viral; D006046:Gold