In traditional immuno-polymerase chain reaction (immuno-PCR), a single antibody recognition event is associated with one to three DNA tags, which are subsequently amplified by PCR. Here we describe a nanoparticle-amplified immuno-PCR (NPA-IPCR) assay that combines antibody recognition of enzyme-linked immunosorbent assay (ELISA) with a 50-fold nanoparticle valence amplification step prior to tag amplification by PCR. The assay detects a respiratory syncytial virus (RSV) surface protein using an antibody bound to a 15-nm gold nanoparticle cofunctionalized with thiolated DNA complementary to a hybridized 76-base tag DNA with a tag DNA/antibody ratio of 50:1. The presence of virus particles triggers the formation of a "sandwich" complex composed of the gold nanoparticle construct, virus, and an antibody-functionalized magnetic particle used for extraction. After extraction, DNA tags are released by heating to 95°C and detected via real-time PCR. The limit of detection of the assay was compared with ELISA and reversion transcription (RT) PCR using RSV-infected HEp-2 cell extracts. NPA-IPCR showed an approximately 4000-fold improvement in the limit of detection compared with ELISA and a 4-fold improvement compared with viral RNA extraction followed by traditional RT-PCR. NPA-IPCR offers a viable platform for the development of early-stage diagnostics requiring an exceptionally low limit of detection.
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