Characterization and comparative performance of lentiviral vector preparations concentrated by either one-step ultrafiltration or ultracentrifugation

Quantitative real-time single particle analysis of virions

“…Methods to determine infection levels include measurements of cytopathic effects such as plaque forming and 50% tissue culture infectious dose (TCID 50 ) assays but also flow cytometric measurements of cellular transduction after infection with viral particles carrying reporter genes such as green fluorescent protein (Metzner et al, 2008;Papanikolaou et al, 2013). While hemagglutination assays directly measure the propensity of viral proteins to crosslink susceptible cell types, serological methodologies also measure the presence of viral antigens, albeit indirectly, by determining antibody conversion.…”

“…Recently, technical progress in the field of microscopy as well as the adaptation of applications originally developed for use in nanotechnology crossed over to uses in virology and made the quantitative analysis of single viral particles as physical entities more feasible. Technologies include atomic force microscopy (AFM) (Ohnesorge et al, 1997), laser light scattering applications such as multiple-angle laser light scattering (MALLS) (Bousse et al, 2013;Wei et al, 2007) or nanoparticle tracking analysis (NTA) (Papanikolaou et al, 2013;Filipe et al, 2010;Kramberger et al, 2012;Anderson et al, 2011;Du et al, 2010), tunable resistive pulse sensing (TRPS, a method based on the Coulter principle) (Vogel et al, 2011;Farkas et al, 2013;Rybakova et al, 2013), and flow cytometry (FC) variants (Brussaard et al, 2000;Ferris et al, 2011;Stepp et al, 2011Stepp et al, , 2010Kemp et al, 2012). Other methods that appear to fall into this category are not discussed in any greater detail, such as viral quantitative capillary electrophoresis (vqCE) (Mironov et al, 2011), since correlates of particle counts (such as nucleic acid amounts) are used for calculation of virus titers, similar to qPCR, rather than the presence of virion particles.…”

“…When using ensemble methods, subpopulations may be hidden, indicating the importance of single particle analysis. transduction and NanoSight, only 0.1% of nascent virions was found to be infective (Papanikolaou et al, 2013) (see also Table 3), providing important information on the quality of the vector preparation. In addition, NTA total particle titers of fluorescently tagged non-infectious HIV-1 virus-like particles were shown to be in good agreement with measurements based on electron microscopy and p24 ELISA (1.24 Â 10 11 ; 1.14 Â 10 11 and 1.30 Â 10 11 , respectively) (Gutierrez- Cervera et al, 2013).…”

“…VC uses two separate fluorescent dyes to stain virus samples: one specific for proteins, the other for nucleic acids. Both, a certain size of virus (425 nm) and a certain length of viral genome (49000 nt/bp) are necessary to guarantee a sufficient level of staining for For information on pricing please contact manufacturer References Bousse et al, 2013;Wei et al, 2007;Chuan et al, 2008;Pease et al, 2009Papanikolaou et al, 2013Kramberger et al, 2012;Du et al, 2010;Gutierrez-Granados et al, 2013;Cervera et al, 2013;Cayatte et al, 2013Ferris et al, 2011Stepp et al, 2011Stepp et al, , 2010Kemp et al, 2012Vogel et al, 2011Farkas et al, 2013;Rybakova et al, 2013;Brussaard et al, 2000 a According to manufacturer. b According to Bousse et al (2013).…”

Continuous separation of protein loaded nanoparticles by simulated moving bed chromatography

“…Moreover, at the time more sophisticated nanoparticles with different characteristics are produced with open applications to different fields ranging from agriculture such as herbicide [1,2] to medicine such as vaccines, cancer treatment and drug delivery [3–9]. At the moment the separation of protein loaded nanoparticles is mostly done by ultracentrifugation [10] if done at all [9]. Some papers reported counter current separation of different kinds or sizes of nanoparticles, but didn’t account for special needs of protein-nanoparticle separation and are dependent on specific interactions of the nanoparticle and the chromatography material [11].…”