Simultaneous purification of influenza haemagglutinin and neuraminidase proteins by immunochromatography.
A new and rapid method for co-purification of haemagglutinin (HA) and neuraminidase (NA) proteins from influenza A/H3N2 viruses is described. Surface glycoproteins were first solubilized using a non-ionic detergent under high ionic strength conditions, then they were separated by chromatography on sepharose previously bound to monoclonal antibodies (MAbs) directed either against HA (IaH-chromatography) or against NA (IaN-chromatography). Depending on the protein specificity of the MAb immobilized on the column, HA or NA was bound to sepharose and the counterpart protein was free in the flow-through volume. IaH-chromatography and IaN-chromatography proved equally efficient in term of recoveries (> 75%) and purity (> or = 99%) of both HA and NA but differences appeared when considering functional and antigenic properties of pure proteins. Those properties were highly retained in IaH- and IaN-derived HA as well as in IaH-derived NA while IaN-NA was partially degraded. IaH-chromatography allowed the co-purification of HA and NA proteins in heterologous antigen-antibody system with a 50% rate of cross reactivity. IaH-HA and IaH-NA may be suitable for immunity studies, standardization of influenza vaccine and for diagnostic purposes.
Gerentes
L
L
Laboratoire de Virologie-Faculté de Médecine, Centre National de Référence de la Grippe, Lyon, France.
Kessler
N
N
Thomas
G
G
Aymard
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000302:isolation & purification; D000956:Antigens, Viral / Q000302:isolation & purification*; D002845:Chromatography / Q000379:methods*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000302:isolation & purification*; D006801:Humans; D009980:Influenza A virus / Q000201:enzymology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009439:Neuraminidase / Q000302:isolation & purification*; D011817:Rabbits; D012685:Sepharose
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D000956:Antigens, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012685:Sepharose; D009439:Neuraminidase