title: Trypsin pre-treatment corrects SRID over-estimation of immunologically active, pre-fusion HA caused by mixed immunoprecipitin rings.
abstract: Influenza vaccines are the primary intervention to prevent the substantial health burden of seasonal and pandemic influenza. Subunit and split influenza vaccines are formulated, released for clinical use, and tested for stability based on their content of immunologically active (capable of eliciting functional antibodies) hemagglutinin (HA). Single-radial immunodiffusion (SRID), the standard in vitro potency assay in the field, is believed to specifically detect immunologically active HA. We confirmed that, with conformationally homogeneous HA preparations, SRID specifically detected native, pre-fusion HA, which elicited influenza neutralizing and hemagglutination inhibiting (HI) antibodies in mice, and it did not detect low-pH stressed, post-fusion HA, which was selectively removed from the SRID gel during a blotting step and was significantly less immunologically active. This selective detection was due to the SRID format, not a conformational specificity of the sheep antiserum used in the SRID, as the same antiserum detected non-stressed and low-pH stressed HA similarly when used in an ELISA format. However, when low-pH stressed HA was mixed with non-stressed HA, SRID detected both forms in mixed immunoprecipitin rings, leading to over-quantification of pre-fusion HA. We previously reported that trypsin digestion of antigen samples selectively degrade stressed HA, so that an otherwise conformationally insensitive biophysical quantification technique, reversed-phase high pressure liquid chromatography (RP-HPLC), can specifically quantify trypsin-resistant, immunologically active, pre-fusion HA. Here, we report that trypsin digestion can also improve the specificity of SRID so that it can quantify immunologically active, pre-fusion HA when it is mixed with less immunologically active, post-fusion HA.
authors.affiliation: Seqirus, A CSL Company, 45 Sidney Street, Cambridge, MA 02139, USA.
authors.forename: Yingxia
authors.initials: Y
authors.lastname: Wen
authors.affiliation: Seqirus, A CSL Company, 45 Sidney Street, Cambridge, MA 02139, USA.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Palladino
authors.affiliation: Seqirus, A CSL Company, 45 Sidney Street, Cambridge, MA 02139, USA.
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Seqirus, A CSL Company, 45 Sidney Street, Cambridge, MA 02139, USA.
authors.forename: Annette
authors.initials: A
authors.lastname: Ferrari
authors.affiliation: Seqirus, A CSL Company, 45 Sidney Street, Cambridge, MA 02139, USA.
authors.forename: Xiuwen
authors.initials: X
authors.lastname: Ma
authors.affiliation: Seqirus, A CSL Company, 45 Sidney Street, Cambridge, MA 02139, USA.
authors.forename: Liqun
authors.initials: L
authors.lastname: Han
authors.affiliation: Seqirus, A CSL Company, 45 Sidney Street, Cambridge, MA 02139, USA.
authors.forename: Philip R
authors.initials: PR
authors.lastname: Dormitzer
authors.affiliation: Seqirus, A CSL Company, 45 Sidney Street, Cambridge, MA 02139, USA. Electronic address: ethan.settembre@seqirus.com.
authors.forename: Ethan C
authors.initials: EC
authors.lastname: Settembre
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007252:Influenza Vaccines / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D009976:Orthomyxoviridae Infections / Q000517:prevention & control; D012680:Sensitivity and Specificity; D012756:Sheep; D014357:Trypsin / Q000737:chemistry*; D064166:Vaccine Potency
chemical_list: D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D014357:Trypsin
keywords: Hemagglutinin; Influenza; Potency; Single-radial immunodiffusion; Trypsinization; Vaccine