Exosome specific biomarkers

EVerZom provides exosome characterization services to measure specific biomarkers like tetraspanins but many others.

Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS, Leprechaun, Unchained Labs)

SP-IRIS is an automated exosome analyzing instrument at the single-particle level. EVs are captured on a chip containing spots coated with anti-tetraspanin antibodies (TSP CD63, CD81, CD9). Captured EVs are detected with a panel of 3 fluorescent antibodies. This technique allows specific quantification of TSP+ EVs, even at a diameter <50nm due to fluorescence, and colocalization of markers. However, no data is provided on TSP- particles, which do not attach to the chips. It is also possible to tailor the chips and the detection antibodies to quantify any surface or cargo protein markers. Required EV quantity is minimal, around 5×10^6 EVs. 

Below is our template for presenting SP-IRIS results for a batch of EVs produced from adipose tissue-derived MSCs, purified by TFF.

ELISA

ELISA allows capturing, detecting, and quantifying EV in both human fluids and cell culture supernatants (Logozzi & al, 2009). The method is able to provide a precise quantification of EV protein markers or cargo, simultaneously in different samples.

Western Blot

Isolated EV can be characterized by western blotting, a widely used technique to detect specific
protein marker or cargo in a sample or even to indirectly semi-quantify EVs by
the total amount of exosomal proteins in a preparation. According to MISEV2018
guidelines, it is necessary to demonstrate the presence of both a transmembrane
protein (tetraspanins) and a cytosolic protein (TSG101,…) to confirm EV
presence.

MACSPlex exosome kit (Miltenyi)

This is a multiplexed flow cytometry assay which provides a relative quantification of 37 exosomal surface epitopes simultaneously. It is based on a cocktail of fluorescently labeled beads, which can be dinstinguished by flow cytometry, each of them coupled to a specific antibody binding exosomal surface epitope. Exosomes are then stained in sandwich with anti-tetraspanins antibodies, which allows the quantification level during measurement.

Fluorescent associated Nanoparticle tracking Analysis (NTA)

NTA is the main technique for EV concentration and sizing. Fluorescent staining of EV markers or cargo can improve NTA sensitivity for small particles, and help to discriminate with non-EV particles. Optimized with CD63 at EVerZom, it is possible to develop the staining for any markers.

Luminex

Luminex is a flow based system enabling an accurate analysis of EV. It consists of pre-coated beads loaded with analyte capture antibodies then, a second analyte-specific antibody is added to the mixture to allow the detection of positive beads by a specific flow cytometry instrument. This technique, which is based on the same general principle than an ELISA, permits to mix at the beginning different beads, targeting different analytes in your solution. Therefore, Luminex permits to analyze simultaneously different analytes with a lot of commercial or customized kit with generally the same accuracy than ELISA and a better sensitivity.

qPCR

EV, exosomes and microvesicles, have been shown to contain potential RNA

such as miRNA or mRNA that may be used in the diagnosis of various biomarkers. They
are also a vehicle of choice for charging therapeutic nucleic acid (siRNA,
plasmid DNA…). Quantitative Polymerase-Chain-Reaction (qPCR) is the best method
to quantify a specific DNA or RNA (if coupled to a reverse transcription)
sequence from an EV sample after extraction. qRT-PCR allows confirming or not
the presence of a RNA/DNA of interest in EV preparation, or comparing the
efficiency of loading in EV between preparations.