title: Application of deglycosylation to SDS PAGE analysis improves calibration of influenza antigen standards.
abstract: Each year the production of seasonal influenza vaccines requires antigen standards to be available for the potency assessment of vaccine batches. These are calibrated and assigned a value for haemagglutinin (HA) content. The calibration of an antigen standard is carried out in a collaborative study amongst a small number of national regulatory laboratories which are designated by WHO as Essential Regulatory Laboratories (ERLs) for the purposes of influenza vaccine standardisation. The calibration involves two steps; first the determination of HA protein in a primary liquid standard by measurement of total protein in a purified influenza virus preparation followed by determination of the proportion of HA as determined by PAGE analysis of the sample; and second, the calibration of the freeze-dried reference antigen against the primary standard by single radial immunodiffusion (SRD) assay. Here we describe a collaborative study to assess the effect of adding a deglycosylation step prior to the SDS-PAGE analysis for the assessment of relative HA content. We found that while the final agreed HA value of the samples tested was not significantly different with or without deglycosylation, the deglycosylation step greatly improved between-laboratory agreement.
authors.affiliation: Division of Virology, National Institute for Biological Standards and Control, Health Protection Agency, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom.
authors.forename: Ruth
authors.initials: R
authors.lastname: Harvey
authors.forename: Michelle
authors.initials: M
authors.lastname: Hamill
authors.forename: James S
authors.initials: JS
authors.lastname: Robertson
authors.forename: Philip D
authors.initials: PD
authors.lastname: Minor
authors.forename: Galina M
authors.initials: GM
authors.lastname: Vodeiko
authors.forename: Jerry P
authors.initials: JP
authors.lastname: Weir
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Takahashi
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Harada
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Itamura
authors.forename: Pearl
authors.initials: P
authors.lastname: Bamford
authors.forename: Tania
authors.initials: T
authors.lastname: Dalla Pozza
authors.forename: Othmar G
authors.initials: OG
authors.lastname: Engelhardt
mesh_terms: D000956:Antigens, Viral / Q000737:chemistry*; D002138:Calibration; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods / Q000592:standards*; D006031:Glycosylation; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry*; D053118:Influenza A Virus, H1N1 Subtype / Q000737:chemistry*; D053122:Influenza A Virus, H3N2 Subtype / Q000737:chemistry*; D007252:Influenza Vaccines / Q000737:chemistry*; D014944:World Health Organization
chemical_list: D000956:Antigens, Viral; C543345:H1N1 virus hemagglutinin; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines