In vivo biotinylated recombinant influenza A virus hemagglutinin for use in subtype-specific serodiagnostic assays

There is an urgent need for robust subtype-specific serological tests to diagnose influenza A virus infections in poultry and mammals, including humans

Alexander Postel; Tobias Letzel; Frederik Müller; Ralf Ehricht; Philippe Pourquier; Malte Dauber; Christian Grund; Martin Beer; Timm C. Harder

2010

Scholarcy highlights

  • There is an urgent need for robust subtype-specific serological tests to diagnose influenza A virus infections in poultry and mammals, including humans
  • A bigenic pFastBacDual baculovirus transfer vector allowing efficient in vivo biotinylation of soluble HA homo-oligomers expressed via the secretory pathway was developed
  • An Avi-Tag allowed site-specific biotinylation by a coexpressed genetically modified BirA biotin ligase retained in the endoplasmic reticulum
  • Seroreactive mono-biotinylated HA of recent H5 and H7 influenza A subtypes was secreted from recombinant baculovirus infected High-Five insect cells at levels sufficient to directly load streptavidin-coated enzyme-linked immunosorbent assay matrices, thereby avoiding any purification steps
  • The recombinant antigens retained authentic antigenicity, including conformation-dependent epitopes involved in hemagglutination inhibition as detected by monoclonal antibodies. This is the first bigenic in vivo biotinylation system established for use in insect cells with secretable recombinant membrane proteins biotinylated by an ER-retained variant of BirA biotin ligase
  • The proposed technique is expected to significantly increase flexibility in the design of subtype-specific assays, thereby expanding the power of influenza A virus serodiagnosis

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