Complete Characterization of Extracellular Vesicles

EV COunt

Count the number of antigen positive EVs direct from sample with no purification. 

EV SIZE

Measure with high resolution, the size of individual antigen positive EVs

PURIFICATION NOT REQUIRED

Measure the changes in your sample, not the biases from your purification technique. 

MULTIPLEXED SAMPLE ANALYSIS

Capture can characterize multiple populations of EVs from a single sample using many surface markers

STREAMLINED WORKFLOW 

Automated instrument measurement and paralleled sample preparation reduce hands-on time and sample throughput 

VALIDATED ASSAYS

Range of validated capture antibodies available. Custom design (direct or via secondary antibodies) also available 


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PURIFICATION NOT REQUIRED

Variability and biases introduced through sample purification are commonly cited as the largest limiting factor in the progression of EV-based platforms into clinical diagnostics and therapeutics.

It is commonly accepted that the methods used in sample collection and isolation can significantly influence the downstream analysis of vesicles with regards to their function, content, physical form, and count.  Soluble proteins, protein aggregates, lipoproteins, cell organelles, and viruses are candidates for co-purification. Common nanoparticle characterization techniques, such as NTA and TRPS, are unable to discriminate particles that co-purify and are subject to errors in data interpretation.  These co-purified particles influence EV counts that are measured and limit the ability to link clinical observations to measured data. 

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For example, when purifying blood plasma by ultracentrifugation, the majority of detectable particles measured within a sample were found to be lipoproteins rather than EVs in a publication by Sódar et al. 2016. These contaminants make it difficult to relate experimental or clinical changes to measured data. Rare events present in biological samples make the presence of contaminants even more challenging. Non-specific techniques are confounded by contaminants as well as by EVs that are not of interest

ExoView bypasses these challenges by measuring only specific EVs that exhibit target surface antigens. It does so without the need for sample purification. The platform works with as little as 35µL of sample and provides EV phenotype, size and count direct from biological fluids onto a functionalized array without the need for purification. EVs have been measured directly from the following samples without the need for ANY purification:

  • Cell culture with or without bovine EVs (no cross reactivity to human antibodies on capture array)

  • Blood plasma

  • Blood serum

  • Cerebrospinal fluid (CSF)

  • Saliva

  • Follicular fluid

  • Synovial fluid


See ExoView in real-time

This video shows vesicles binding to the ExoView array in real time. The EVs bind to the surface via an antibody/antigen interaction and therefore requires no labeling and no purification of the EV sample. Once bound, the size and count of antigen positive EVs are measured.


COMMON ISSUES WITH PURIFICATION

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MICROARRAY DESIGN

ExoView has been designed specifically to address the needs of EV researchers. The microarray format allows for many probes to be measured simultaneously, dramatically increasing throughput. The ExoView Tetraspanin Kit contains arrays that have been functionalized with antibodies against CD9, CD63, CD81, CD41a plus IgG negative control. Additionally, customers can work with NanoView Biosciences to design arrays with antibodies against an antigen of their choice.


MEASURING EV SIZE

ExoView™ uses Single Particle Interferometric Reflectance Imaging Sensing (SP-IRIS) to measure the size of single EVs with high precision by analyzing the enhanced interferometric signal of bound EVs when attached to a layered substrate.  Using this methodology, EVs as small as 50nm can be imaged and measured. The enhanced interferometric signal has a radius^3 relationship between size and signal intensity that provides excellent resolving capabilities when compared to existing techniques such as nanoparticle tracking analysis (NTA).  EV preparations often exhibit a broad range of sizes and sub-populations, therefore sizing resolution can impact how well a technology can accurately characterize EV samples. ExoView has significantly stronger peak-to-peak resolution and can more accurately resolve distinct vesicle sub-populations within a heterogeneous sample. When using NTA, EVs with a size difference of 2X will result in a 2X difference in scattering, whereas ExoView achieves an 8X difference.  Furthermore, unlike NTA, the presence of larger EVs does not obscure or bias the relative count of particles within a heterogeneous sample population.

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ExoView has unrivaled peak-to-peak size resolution as demonstrated by its ability to resolve five particle populations of differently sized NIST certified polystyrene beads (40nm, 70nm, 100nm, 150nm, and 200nm) within a single measurement.  While polystyrene beads are not a good analog for EVs, the experiment serves to demonstrate the inherent resolving capabilities of the technique. 

NIST particles (50nm, 100nm, 150nm, and 200nm) and their specified distrubtions. The solid blue line is the ExoView results for a 4 particle mixed sample.

NIST particles (50nm, 100nm, 150nm, and 200nm) and their specified distrubtions. The solid blue line is the ExoView results for a 4 particle mixed sample.


MEASURING EV COUNT

The number of EVs bound to a chip can be measured and related to a concentration of antigen-positive EVs in solution. Control tests are performed to ensure that the binding events measured are specific and not related to non-specific binding or aggregates associated with the antibody used to functionalize the chip surface. The array is measured prior to sample incubation and any contaminant events can be excluded from the analysis. Similarly, non-specific binding events observed on the negative control can be subtracted from experimental results.

The graph below shows the linearity response of the ExoView instrument. As a measure of comparability, a sample of EVs was measured using an NTA instrument. The sample has been linearly diluted and measured using the ExoView platform and the number of CD81-positive EVs were counted at a range of dilutions.