Hi, my name is Struan, and I am a scientist at Izon Science.
In this video I will be discussing Izon’s qEV concentration kit,
including its principles,
considerations for obtaining the best outcomes,
and suggested protocols.
Some EV isolation methods enrich EVs, but largely compromise EV purity.
However, Izon’s qEV Columns provide a rapid, reliable,
and standardisable system for EV isolation and importantly,
this is done while providing a high level of EV purity.
That is why qEV columns have become very popular among EV researchers and developers of EV diagnostics.
However, despite having purified EV preparations,
a higher concentration of pure EVs may be required for certain downstream applications.
Therefore, a post-purification concentration step may be needed.
There are a few methods used to concentrate EV samples.
Ultrafiltration is a common concentration technique,
but it can be imprecise as it relies on broad particle size-based separation that is not designed specifically for EVs.
Also, ultrafiltration devices have a fixed minimal retrievable volume, with limited concentration potential.
And, EV loss can occur when EVs adhere to the filter.
Ultracentrifugation-based concentration methods might damage EV integrity,
leading to a loss of EVs and their molecular cargo.
Where efficient and specific enrichment and concentration methods are needed for purified EV samples,
Izon has fulfilled this niche with the launch of the qEV Concentration Kit.
The qEV Concentration Kit is based on Ceres Nanosciences Nanotrap® Extracellular Vesicle Particles.
Nanotrap EV particles are hydrogel particles of approximately 260 nanometres in diameter,
they are made of thermostable cross-linked polymers which have been shown to retain smaller target analytes in variable temperatures or pH conditions.
Importantly, the surface of Nanotrap® EV Particles have been functionalised with a specific chemical affinity bait
that has shown a significantly higher affinity for EVs compared to other similar biological nanostructures.
This western blot data shows the detection of EV markers Alix and CD63 in tissue culture supernatant,
following the use of Nanotrap particles.
The qEV Concentration Kit is an all-in-one system for capturing and concentrating intact EVs isolated using any of the qEV Columns.
Using the qEV Concentration Kit,
EVs can be concentrated to a customisable pellet,
which can be used as it is for maximal EV enrichment or resuspended in a desired volume and buffer type.
The kit does not require special equipment,
and does not use ultra-speed centrifugation,
precipitation reagents or protease treatments.
Nanotrap® EV Particles are designed to irreversibly bind to the EVs,
meaning that there is no elution step available to detach intact EVs.
An EV lysis step can be included as part of the downstream assay, to release EV-associated cargo.
Therefore, the kit is not suited to downstream analyses that quantify EVs as free particles.
Instead, the qEV Concentration Kit is best suited to capturing and concentrating purified EVs prior to the detection,
quantification or molecular characterisation of low-abundance specific EV markers or molecules.
For example, this could be for studies assessing RNA or proteins in EVs.
This figure here shows results from a Reverse Transcription qPCR study of mRNA targets in plasma EVs concentrated with the qEV Concentration kit compared with Amicon filters.
There are a few things you might want to consider when planning studies involving the qEV Concentration kit.
Alcohol-based lysis buffers that might be used for some of the applications decrease lysis efficiency of the Nanotrap® EV Particle-bound EVs.
Therefore, alcohol-free lysis buffers are recommended at the EV lysis step of any downstream assay.
Again, keep in mind that Nanotrap EV particles cannot be separated from intact EVs.
As always, be sure to include appropriate controls so you can assess potential background effects or interference in your experiment.
Another important thing to note is that EV binding sites are not saturated when bound to Nanotrap® EV Particles.
This is relevant if they are required elsewhere, for example in other tagging or staining assays.
However, it is a good idea to include your own tagging or staining controls with Nanotrap® EV Particles to determine the interference with signal intensity.
The qEV concentration kit comes in a box which holds a 5 mL container of Nanotrap® EV Capture Particles.
In the box you will also find information about storage recommendations.
The qEV concentration kit uses a very simple and quick protocol.
The first step is to pool the qEV purified collection volume of interest containing pure EVs.
In this table, we can see PCVs from a range of qEV columns.
The second step involves adding the appropriate volume of Nanotrap® EV Capture Particles to the pooled purified collection volume.
The volume of Nanotrap® EV Capture Particles required to concentrate EV-containing samples will depend on the sample volume,
as shown in the bottom table.
Next, the mixture is incubated with rotation for one hour,
to ensure efficient and specific binding of EVs to Nanotrap® EV particles.
In step 4,
the mixture must be centrifuged at 16,800 g for 10 minutes to pellet the EVs bound to Nanotrap® EV Particles.
The fifth step involves removing the supernatant,
being careful not to disturb the pellet containing EVs bound to Nanotrap® EV Particles.
And finally, the pellet is ready for downstream applications.
The pellet can be resuspended in a desired buffer volume or used directly with a method-appropriate lysis buffer,
to avoid further dilution of concentrated EVs.
Overall, the qEV concentration kit provides a way to concentrate EVs following qEV isolation,
to enable analysis downstream.
Example protocols for suitable downstream assays are provided in the user manual.
These suggested protocols are a guide to help obtain the best results from EVs concentrated using the qEV Concentration kit,
for Western blot and mass spec,
and for RNA extraction using the qEV RNA Extraction kit,
a highly sensitive kit free from phenol and chloroform.
Thanks for watching! For resources regarding the qEV concentration kit and other related products,
be sure to visit izon.com.