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Methods Mol Biol ; 1026: 93-110, 2013.
Article in English | MEDLINE | ID: mdl-23749572

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.


Subject(s)
Gold/chemistry , Immunoassay/methods , Metal Nanoparticles/chemistry , Real-Time Polymerase Chain Reaction/methods , Respiratory Syncytial Viruses/isolation & purification , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Cell Line , DNA, Viral/genetics , DNA, Viral/isolation & purification , Limit of Detection , Magnets/chemistry , Microspheres , Quartz Crystal Microbalance Techniques , RNA, Viral/genetics , RNA, Viral/isolation & purification , Reproducibility of Results , Respiratory Syncytial Viruses/immunology
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