title: Development of an enzyme-linked immunoassay for the quantitation of influenza haemagglutinin: an alternative method to single radial immunodiffusion.
abstract: BACKGROUND
  The current method used to measure haemagglutinin (HA) content for influenza vaccine formulation, single radial immunodiffusion (SRID), is lengthy and relies on the availability of matched standardised homologous reagents. The 2009 influenza pandemic highlighted the need to develop alternate assays that are able to rapidly quantitate HA antigen for vaccine formulation.
  
  
  OBJECTIVE
  The aim of this work was to develop an enzyme-linked immunoassay (EIA) for the rapid quantitation of H1, H3, H5 and B influenza HA antigens.
  
  
  METHODS
  Monoclonal antibodies (mAbs) selected for haemagglutination inhibition (HAI) activity were conjugated with horseradish peroxidase and used to establish a capture-detection EIA for the quantitation of HA antigen. Results were compared with the appropriate reference SRID assays to investigate assay performance and utility.
  
  
  RESULTS
  Quantitation of HA antigen by EIA correlated well with current reference SRID assays. EIA results showed equivalent precision and exhibited a similar capacity to detect HA antigen in virus samples that had been used in either stability or splitting studies, or subjected to physical or chemical stresses. EIA exhibited greater sensitivity than SRID and has the potential to be used in high-throughput applications.
  
  
  CONCLUSIONS
  We demonstrated the utility of EIA as a suitable alternative to SRID for HA antigen quantitation and stability assessment. This approach would lead to earlier availability of both seasonal and pandemic vaccines, because of the extended cross-reactivity of reagents.
authors.affiliation: CSL Ltd, Parkville, Vic., Australia.
authors.forename: Jesse
authors.initials: J
authors.lastname: Bodle
authors.forename: Erin E
authors.initials: EE
authors.lastname: Verity
authors.forename: Chi
authors.initials: C
authors.lastname: Ong
authors.forename: Kirsten
authors.initials: K
authors.lastname: Vandenberg
authors.forename: Robert
authors.initials: R
authors.lastname: Shaw
authors.forename: Ian G
authors.initials: IG
authors.lastname: Barr
authors.forename: Steven
authors.initials: S
authors.lastname: Rockman
mesh_terms: D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000032:analysis*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D005779:Immunodiffusion / Q000379:methods; D007252:Influenza Vaccines / Q000737:chemistry* / Q000592:standards*; D013678:Technology, Pharmaceutical / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D006735:Horseradish Peroxidase