Rapid and specific influenza virus detection by functionalized magnetic nanoparticles and mass spectrometry

Abstract

BackgroundThe timely and accurate diagnosis of specific influenza virus strains is crucial to effective prophylaxis, vaccine preparation and early antiviral therapy. The detection of influenza A viruses is mainly accomplished using polymerase chain reaction (PCR) techniques or antibody-based assays. In conjugation with the immunoassay utilizing monoclonal antibody, mass spectrometry is an alternative to identify proteins derived from a target influenza virus. Taking advantage of the large surface area-to-volume ratio, antibody-conjugated magnetic nanoparticles can act as an effective probe to extract influenza virus for sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and on-bead mass spectrometric analysis.ResultsIron oxide magnetic nanoparticles (MNP) were functionalized with H5N2 viral antibodies targeting the hemagglutinin protein and capped with methoxy-terminated ethylene glycol to suppress nonspecific binding. The antibody-conjugated MNPs possessed a high specificity to H5N2 virus without cross-reactivity with recombinant H5N1 viruses. The unambiguous identification of the captured hemagglutinin on magnetic nanoparticles was realized by SDS-PAGE visualization and peptide sequence identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS).ConclusionsThe assay combining efficient magnetic separation and MALDI-MS readout offers a rapid and sensitive method for virus screening. Direct on-MNP detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provided high sensitivity (~103 EID50 per mL) and a timely diagnosis within one hour. The magnetic nanoparticles encapsulated with monoclonal antibodies could be used as a specific probe to distinguish different subtypes of influenza.

In vivo monitoring of rat macrophages labeled with poly(l‐lysine)‐iron oxide nanoparticles

“…1 Biomedical applications benefit from the fact that the superparamagnetic particles can be easily manipulated and/ or detected using an external magnetic field. The applications using manipulations include immunoassays, 2 separation of microorganisms and biomolecules, such as viruses, 3 hormones, 4 oligonucleotides, 5 DNA, 6 proteins 7,8 and cells, 9 targeted drug and gene delivery. 10 However, the use of particles for detection has concentrated on cell tracking, [11][12][13] tagging and imaging [by magnetic resonance imaging (MRI)] 14 as well as on cancer treatment by hyperthermia.…”

Beads-Based Electrochemical Assay for the Detection of Influenza Hemagglutinin Labeled with CdTe Quantum Dots

“…The band at 38 kDa represents the hemagglutinin fragment from influenza B virus. The band at 48 kDa was assigned to the glycosylated HA [30]. The band at 56 kDa is connected with the presence of nucleocapsid protein (NP).…”

“…The other two conditions optimized directly included the labeled HA isolation reaction. Tested conditions included six temperatures (5,25,35,45, 55 and 65 °C) and reaction times (5,10,15,20,30,45 and 60 min). From the results it follows that best signals were obtained at temperatures higher than 35 °C and the temperature of 45 °C was established as the best, based on the Cd peak results (blue columns in Figure 4B) and HA peak (red rhombi in Figure 4B), which correspond to the range of body temperatures of humans and small animals, especially birds, which serve as influenza reservoirs [32].…”

Inhibitory activity of selenium nanoparticles functionalized with oseltamivir on H1N1 influenza virus

“…9,10 Neuraminidase (NA) and HA are the most significant glycoproteins on the surface of the influenza virus. 11,12 NA plays an important role in assisting the virus cleave the linkage between sialic acid and HA, when mature viruses separate from the host cell surface. 13 Vaccination is a common method of restraining the spread of influenza infections.…”