Tracking Aggregation of Virus-Like Particles Using Dynamic Light Scattering

초록

Source: Moore, M. D., et al. Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity. J. Vis. Exp. (2017)

The video demonstrates a protocol for monitoring the heat-induced aggregation of virus-like particles using dynamic light scattering (DLS). It begins with preparing a monodisperse suspension of purified particles. The sample is then loaded into a preheated DLS instrument. As the capsid proteins denature with heat, exposed hydrophobic regions cause particle aggregation. DLS detects changes in particle size over time, enabling real-time tracking of the aggregation process.

프로토콜

1. DLS for Detecting Virus Aggregation in Real Time

NOTE: The steps below may be specific to the software and instrument used, but can be adapted and applied to similar devices.

1. Create a new size SOP in the Dynamic Light Scattering (DLS) instrument software using: Material = Protein, Dispersant = Phosphate-buffered saline (PBS), Temperature = as desired, Equilibration time = 0 sec, Cell type = quartz cuvette, Measurement angle = 173° Backscatter, Measurement duration = automatic, Number of measurements = 50 (may be increased or decreased depending on aggregation rate), Delay between measurements = 0 sec, Data processing: Analysis model = Multiple narrow modes (high resolution). Use the default settings for all other parameters.
2. Select the run arrow within the software to open and name a new sample, and allow the instrument to reach the temperature equilibrium.
3. Obtain purified human norovirus major capsid protein (VP1) of GII.4 Sydney (Accession: JX459908) assembled in capsids.
   NOTE: Capsids in the study were obtained courtesy of R. Atmar (Baylor College of Medicine, Houston, TX).
4. Suspend the Virus-like particles (VLPs) in 1× phosphate-buffered saline buffer (PBS) in a microcentrifuge tube at a concentration of 5 µg/mL and a volume between 200 - 500 µL. Vortex the suspension.
5. Spin down the VLP suspension for 5 - 10 s in a tabletop centrifuge to de-gas. This helps to prevent bubble formation during the heat treatment that interferes with size measurements.
6. Transfer the VLP suspension to a small volume quartz cuvette-avoid bubbles and pipette slowly against the wall of the cuvette. After the instrument has reached temperature equilibrium, insert the cuvette into the sample chamber.
7. Wait 10 s for the sample temperature to equilibrate, then start the run. Start a timer at the start of the run. Stop the timer at the end of the first measurement. Take this time as the first time point. Obtain subsequent time points from the measurement times recorded by the software.
8. Start the run, and monitor the correlogram, distribution fit, and expert advice to evaluate the data quality.
   NOTE: The Polydispersity Index (PDI) will not necessarily be less than 0.3 for these measurements as the sample is expected to be polydisperse during aggregation.
   1. Make sure the correlation coefficient is constant and close to, but not more than 1 at short time, and drops off sharply at one or more later times, characteristic of particle size.
   2. Make sure the distribution fit line follows the data points closely.
9. Analyze the size data.
   1. Determine each time point from the difference in time between each measurement and the initial time point. Measurement durations are not all exactly the same.
   2. Using an intensity distribution, record the size of the peaks with the largest areas for each measurement/time point.
   3. Plot the peak diameters in nm vs. time in min.

재료

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이름	회사	카탈로그 번호	댓글
Purified, assembled human norovirus GII.4 capsids
1x PBS (pH 7.2)	Life Technologies	20012-050
Zetasizer ZSP, or similar particle size analysis instrument	Malvern Instruments	N/A
Vortex	Fisher Scientifc	02-215-365
Quartz cuvette	Hellma	104-002-10-40
Small volume disposable cuvette (to reduce VLP use)	Malvern Instruments	ZEN0040