2.6.1. Immunoblot for VLP detection
VLP production was monitored by spot blot: 3 µL of centrifuged cell
culture supernatants (300 g , 5 minutes) were pipetted onto a
nitrocellulose membrane (Vita Scientific, USA) and allowed to dry before
proceeding with the immunoblot assay. Primary antibody incubation was
performed using either the pan-flavivirus mAb 4G2 (1:8,000) (MAB10216,
Merck, USA) or an anti-yellow fever antibody (1:3,000)
(Bio-Manguinhos/Fiocruz, Brazil), followed by incubation with anti-mouse
IgG HRP-conjugated secondary antibody (1:10,000) (A16011, Invitrogen,
USA) and then ECL Prime detection reagent (GE Healthcare, USA). Dengue
envelope protein (Prospec, Israel) or in-house purified VLP standards
(130-190 μg/mL stock) were used as positive controls and to generate
calibration curves for each spot blot. Approximate concentrations of
VLPs were determined by densitometry using the image analysis software
ImageJ (National Institutes of Health, USA).