title: Rapid and specific influenza virus detection by functionalized magnetic nanoparticles and mass spectrometry.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  Iron 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).
  
  
  CONCLUSIONS
  The 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 (~10(3) EID(50) 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.
authors.affiliation: Department of Chemistry, National Taiwan University, Taipei, 106, Taiwan.
authors.forename: Tzu-Chi
authors.initials: TC
authors.lastname: Chou
authors.forename: Wei
authors.initials: W
authors.lastname: Hsu
authors.forename: Ching-Ho
authors.initials: CH
authors.lastname: Wang
authors.forename: Yu-Ju
authors.initials: YJ
authors.lastname: Chen
authors.forename: Jim-Min
authors.initials: JM
authors.lastname: Fang
mesh_terms: D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005290:Ferric Compounds / Q000737:chemistry*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology / Q000302:isolation & purification*; D053125:Influenza A Virus, H5N2 Subtype / Q000276:immunology / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis*; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D000914:Antibodies, Viral; D005290:Ferric Compounds; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D058185:Magnetite Nanoparticles; C000499:ferric oxide
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