Abstract
An enzyme linked immunosorbent assay (ELISA) method was developed to analyze the assembly of a tetravalent mosaic influenza nanoparticle (NP) vaccine, Flumos-v1, consisting of hemagglutinin trimers (HAT) from H1 (A/Idaho/07/2018), H3 (A/Perth/1008/2019), HBV (Vic-B/Colorado/06/2017) and HBY (Yam-B/Phuket/3073/2013) strains. The sandwich ELISA assay used lectin from Galanthus nivalis as a universal capture reagent for all HAT strains and specific monoclonal antibody (mAb) to detect corresponding hemagglutinin antigen. The mAb binding of HATs incorporated into NPs diverged from those for single HAT solutions, resulting in inaccurate quantitation of assembled HATs. An optimized zwittergent treatment was used to fully dissociate the influenza NP and aligned binding activities in each pair of single HAT and dissociated HAT from NP. The dissociated HATs were then quantified against their corresponding HAT standard solutions for three development lots of FluMos-v1 vaccine and the assembly ratio of all four HATs was calculated. The molar ratio of different HATs incorporated into this quadrivalent NP vaccine was consistent and determined as H3:H1: HBV: HBY ∼ 1.00:0.92:0.96:0.87, which was close the expected 1:1:1:1 ratio and confirmed a proper assembling of multivalent NP.
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