Reversed phase monolithic analytical columns for the determination of HA1 subunit of influenza virus haemagglutinin

Abstract

Monoliths are chromatographic stationary phases, which were specially designed for efficient purification of large biomolecules, like proteins, viruses and DNA. In this work, the small scale monolithic butyl (C4) and styrene-divinyl benzene (SDVB) columns were applied for reversed phase analyses of various degraded influenza viruses. The binding of the HA1 subunit of haemagglutinin to the monolithic columns was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blot. The working linear range was determined as 1.60×10(10) viral particles/mL to at least 1.64×10(11) viral particles/mL, the limit of detection was found to be 2.56×10(9) virus particles/mL and the limit of quantification was 5.12×10(9) virus particles/mL. The analytical HPLC method developed with the H1N1 virus was also applicable for the analytics of the HA1 subunit of H3N2 influenza virus and the influenza B virus.