A sensitive adenovirus immunoassay as a model for using nanoparticle label technology in virus diagnostics

Abstract

Currently, PCR-based hybridization assays are widely applied in adenovirus diagnostics. However, the technology requires tedious sample preparation, and the amplification phase is susceptible to various contaminants leading to inconvenient and time-consuming assay procedure. Methods relying on viral antigen detection, e.g. immunofluorometric assays (IFMAs) and enzyme immunoassays (EIAs), are less complicated to carry out, but they provide limited sensitivity. Our aim was to develop a simple and sensitive adenovirus assay based on direct antigen detection via sandwich-forming immunoreaction. The assay employed highly fluorescent europium(III)-chelate-doped nanoparticle labels and selection of high affinity monoclonal antibodies (anti-hexon) coated on label particles and microtitration wells. The extremely high specific activity of the nanoparticle labels enabled the detection limit over 5000 virus particles per millilitre with purified virus particles. The sensitivity was improved by three orders of magnitude (800-fold) compared to concurrent time-resolved IFMA. Furthermore, the nanoparticle assay showed reasonably low coefficients of variation (4.0-20%) and excellent linearity of more than four orders of magnitude (from below 10(5) to 10(9) virus particles per millilitre). Analyzed nasopharyngeal patient specimens revealed a minor disturbance of matrix components, which could be avoided by dilution. The average signal difference between negative and positive samples was nearly four orders of magnitude. The developed assay was sensitive and more convenient approach to adenovirus screening compared to available assays. In addition, the study demonstrates the potential of nanoparticles in sensitive screening of viral analytes.