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.

Detection of <em>Mycobacterium tuberculosis</em> purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

“…The activated thiolated DNA sequence was desalted and purified using centrifugal filters. 13 The purified capture DNA was stored at -20°C until further use.…”

“…Functionalization of GNPs (~20 nm) with antibodies and DNA was performed according to previously described method by Perez et al 12,13 with little modifications. GNPs were used at a concentration of ~1×10 11 particles/mL in 0.1 mM phosphate buffer and pH was adjusted to 9.3 with 1 M NaOH.…”

“…11 In particular, due to small size (1-100 nm), corresponding high surface-to-volume ratio and unusual target binding properties, nanoparticles (NPs) have been used as signal amplification tools. 12,13 One such example is the NP-based bio-barcode amplification assay (BCA), which was initially introduced by Nam et al 14 for protein and nucleic acid detection with ultrahigh sensitivity using multiple types of NPs, such as gold nanoparticles (GNPs), silica, iron oxide and magnetic particles (MPs). Hundreds of barcode DNAs released from each GNP could elicit 2-3 log amplification in detection sensitivity for each captured targets.…”

Detection of potential microbial antigens by immuno-PCR (PCR-amplified immunoassay)

“…In recent years, nanobiotechnology has emerged as the most promising tool for the development of a powerful strategy for drug delivery diagnostics (Griffiths et al , 2010). Besides circumventing the background noise, the use of magnetic beads and nanoparticle-based I-PCR can further improve detection limits and reduce washing steps, as well as reducing incubation steps, thus improving the assay (Adler et al , 2008; Chen et al , 2009; Perez et al , 2013). In the magnetic bead-based I-PCR assay (), the capture antibody is adsorbed to the magnetic beads to capture the antigen (Barletta et al , 2009) and the sandwich I-PCR format is then followed using streptavidin as a bridge between the detection antibody and the reporter DNA.…”

“…The immunocomplexes are heated to release the signal DNA, which is quantified by real-time PCR. In another I-PCR format, known as nanoparticle-amplified I-PCR (NPA-I-PCR) (), antigen is captured using antibody-functionalized magnetic beads (Perez et al , 2011, 2013). The gold nanoparticles are functionalized with the detection antibodies and thiolated DNA complementary to the hybridized tag DNA.…”