Perez Jonas W, Adams Nicholas M, Zimmerman Grant R, Haselton Frederick R, Wright David W
Department of Chemistry, Vanderbilt University, Nashville, TN, USA.
Methods Mol Biol. 2013;1026:93-110. doi: 10.1007/978-1-62703-468-5_8.
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.
早期检测对于有效治疗儿科病毒感染至关重要。在传统免疫PCR中，单个抗体识别事件与一到三个DNA标签相关联，随后通过PCR进行扩增。在本方案中，我们描述了一种纳米颗粒扩增免疫PCR检测方法，该方法将传统ELISA的抗体识别与50倍的纳米颗粒价态扩增步骤相结合，随后通过传统PCR进行扩增。该检测方法使用与15 nm金纳米颗粒结合的Synagis抗体检测呼吸道合胞病毒（RSV）表面融合蛋白，该金纳米颗粒与与杂交的76碱基标签DNA互补的硫醇化DNA共功能化。Synagis与标签DNA的比例为50比1。病毒颗粒的存在触发了由金纳米颗粒构建体、病毒和用于提取的1μm抗体功能化磁性颗粒组成的“三明治”复合物的形成。使用磁铁分离含病毒的复合物，通过加热至95°C释放DNA标签，并通过实时PCR进行检测。使用RSV感染的HEp-2细胞提取物，将纳米颗粒扩增免疫PCR检测方法的检测限与传统ELISA和传统RT-PCR进行了比较。与ELISA相比，纳米颗粒扩增免疫PCR的检测限提高了约4000倍，与传统RT-PCR相比，检测限提高了四倍。纳米颗粒扩增免疫PCR为开发需要极低检测限的早期诊断提供了一个可行的平台。
