title: Monitoring of influenza virus hemagglutinin in process samples using weak affinity ligands and surface plasmon resonance.
abstract: Surface plasmon resonance (SPR) was used to screen the interaction between a variety of affinity ligands and hemagglutinin (HA) from human influenza virus, with the aim of identifying low affinity ligands useful for the development of a rapid bioanalytical sensor. Three sialic acid-based structures and four lectins were evaluated as sensor ligands. The sialic acid-based ligands included a natural sialic acid-containing glycoprotein, human alpha1-acid glycoprotein (alpha1-AGP), and two synthetic 6'-sialyllactose-conjugates, with varying degree of substitution. The interaction of HA with the four lectin-based ligands, concanavalin A (Con A), wheat germ agglutinin (WGA), Maackia amurensis lectin (MAL), and Sambucus nigra agglutinin (SNA), showed a wide variation of affinity strengths. Affinity and kinetics data were estimated. Strong affinities were observed for Con A, WGA, alpha1-AGP, and a 6'-sialyllactose-conjugate with a high substitution degree, and low affinities were observed for MAL and a 6'-sialyllactose-conjugate with low substitution. The main objective, to identify a low affinity ligand which could be used for on-line monitoring and product quantification, was met by a 6'-sialyllactose-ovalbumin conjugate that had 0.6 mol ligand per mol carrier protein. The apparent affinity of this ligand was estimated to be 1.5+/-0.03 microM (K(D)) on the SPR surface. Vaccine process samples containing HA were analyzed in the range 10-100 microg HA mL(-1) and correlated with single-radial immunodiffusion. The coefficient of variation on the same chip was between 0.010 and 0.091.
authors.affiliation: Division of Biotechnology/IFM, Linköping University, SE-581 83 Linköping, Sweden. cfm@ifm.liu.se
authors.forename: Carl-Fredrik
authors.initials: CF
authors.lastname: Mandenius
authors.forename: Ronghui
authors.initials: R
authors.lastname: Wang
authors.forename: Anna
authors.initials: A
authors.lastname: Aldén
authors.forename: Gunnar
authors.initials: G
authors.lastname: Bergström
authors.forename: Sabine
authors.initials: S
authors.lastname: Thébault
authors.forename: Charles
authors.initials: C
authors.lastname: Lutsch
authors.forename: Sten
authors.initials: S
authors.lastname: Ohlson
mesh_terms: D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000276:immunology / Q000378:metabolism; D053122:Influenza A Virus, H3N2 Subtype / Q000737:chemistry / Q000276:immunology; D007252:Influenza Vaccines / Q000096:biosynthesis* / Q000378:metabolism / Q000592:standards; D007700:Kinetics; D037102:Lectins / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism; D009975:Orthomyxoviridae / Q000276:immunology / Q000378:metabolism*; D010047:Ovalbumin / Q000737:chemistry / Q000378:metabolism; D010063:Ovum / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors
chemical_list: D002352:Carrier Proteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D037102:Lectins; D008024:Ligands; D010047:Ovalbumin; D019158:N-Acetylneuraminic Acid