Identify viral vectors with NanoSight Pro F-NTA

Introduction

A common method for detecting and studying cell behavior is fluorescence detection, which can also be applied to characterize much smaller viral vectors. This application note demonstrates how membrane labeling, using lipophilic dyes, can be used to characterize viral vector formulations with NanoSight Pro fluorescence mode.

Viral vectors membrane labeling 

This study demonstrates the use of NanoSight Pro, utilizing Nanoparticle Tracking Analysis (NTA) technology to characterize lentivirus* and MVA* formulations.

General membrane labeling was performed to confirm the presence of particles containing a lipidic membrane. A lipophilic carbocyanine dye DiO (DIOC18(3)), at the concentration of 1mg/ml, was incubated with lentivirus or MVA samples for 15 minutes at room temperature (Figures 1 and 2). Next, both samples were diluted in PBS to an optimum concentration for NTA analysis. Samples were analyzed with NanoSight Pro, equipped with a 488nm laser and 500LP fluorescence filter as the best configuration for DiO excitation and fluorescence signal collection.

Figure 1: Schematic of labeling Lentivirus with DiO

Figure 2: Schematic of labeling MVA with DiO

Data was collected in both light scatter and fluorescence modes for the samples labeled with DiO and the unlabeled controls. The obtained size distributions of both, lentivirus and MVA formulations, are shown in Fig. 3 and 4.

The labeling was highly effective in both cases, as particle brightness in the light scatter mode increased after labeling (Figures 3 and 4, blue and green line), resulting in a shift toward larger particle sizes compared to the unlabeled control (Figures 3 and 4, grey line). This is likely due to the strong affinity of DiO for binding to any lipid-containing compartments, with the dye adhering to the particle surface in large quantities, creating a "dye corona" around the particles.

Figure 3: Size distribution and correlating NTA images of lentivirus labeled with DiO

Figure 4: Size distribution and correlating NTA images of MVA labeled with DiO

In the case of the lentivirus sample, fluorescence was observed across the entire particle size range and concentration (Figure 3). However, this labeling method does not distinguish lentivirus from other particles, such as extracellular vesicles. For MVA, the fluorescence efficiency was lower than for lentivirus, with a noticeable number of unlabeled particles (Figure 4). Since MVA is a less purified sample, many components lack lipid membranes, preventing the dye from attaching to them. 

Summary 

Membrane labeling using DiO is very easy and effective in staining both lentivirus and MVA vector formulations. It is worth noting that membrane labeling is not specific and cannot distinguish viral vectors from the lipidic components/impurities in the sample. 

Further reading

This application is part of a five-part series. Explore the rest of the series to dive deeper into this topic:

• Quick viral vector purity assessment with NanoSight Pro 
• Viral vector stability and storage assessment by NanoSight Pro
• Identify VSV-G positive lentivirus with NanoSight F-NTA

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*Lentivirus and MVA samples along with formulation buffers were kindly supplied by one of Malvern Panalytical’ s collaborators.