Dr Andrea Krumm describes an exquisitely sensitive microplate-based assay
Exosomes are extracellular vesicles (EVs) of endosomal origin. These vesicles contain proteins, lipids and nucleic acids and facilitate intercellular communication between different cell types within an organism. The rapidly expanding EV research ﬁeld beneﬁts tremendously from tools for the detection of exosomes and their content. Tetraspanin proteins such as CD9, CD63 and CD81 with exposed domains are particularly enriched in the exosome membrane and are often used as exosome biomarkers. These tetraspanins show a distinct pattern of prevalence on exosomes in different cell types. Kits based on time resolved fluorescence speciﬁcally analyse the levels of tetraspanin proteins and thereby detect exosomes. The detection method is compatible with cell culture media, urine or blood.
How the assay works
In a first step, biotinylated antibody specific for a Tetraspanin is bound to a streptavidin coated microplate (Fig. 1). After sample addition, the antibody captures the tetraspanin and thus exosomes. Detection is carried out via another tetraspanin-specific antibody, which is additionally labelled with europium. It binds the exosome and europium can be quantified using a microplate reader capable of time resolved fluorescence measurements. The assay kit containing microplate, antibodies and buffers are provided by Cell Guidance Systems.
How the assay is detected
Europium that is coupled to the tetraspanin-specific detection antibody has fluorescent characteristics. As it exhibits long fluorescence lifetime, it is suited to be recorded in a time-resolved manner. This means fluorescence is recorded over a specific period after excitation and not parallel to excitation of the fluorophore. In the time between excitation and emission, the short-lived autofluorescence decays, so that only the fluorescence of europium is measured. Thus, the detection method is also suitable for complex samples such as blood.
The exosome detection method was tested on a BMG Labtech microplate reader using exosomes purified from conditioned cell culture media and diluted to eight samples containing 0.8-100 µg/ml. The expression of CD9, CD63 and CD81 (commonly used surface exosome markers), were measured on exosomes derived from two different cancer cell lines, derived from prostate and colorectal cancer.
Prostate cancer cells exhibited maximum signal when tested for CD83, whereas CD63 and CD9 were less exposed on exosomes purified from prostate cancer cell culture medium (Fig. 2A). In contrast, exosomes from colorectal cancer medium show high amounts of CD63 and CD9 and low CD81 levels (Fig. 2B).
The quantification method delivers a high linearity with a regression coefficient R² >0.995 for all three exosome markers and for both cell lines. The excellent linearity is combined with lowest variation (low error bars) between the replicates (N=3; Fig. 2A). Both linearity and data variation prove the high assay quality arising from a stable assay combined with robust and sensitive measurements of the BMG Labtech microplate reader.
Dr Andrea Krumm is with BMG Labtech
Figure 1 (above) Principle of the time-resolved immunoflourescence exosomes detection assay. Step 1: binding of antibody. Step 2: addition of biological samples. Step 3: detection of europium-labelled antibody by time-resolved fluorescence.
Figure 2 (above) Marker detection on exosomes from prostate cancer calles (A) or colorectal cancer vells (B). CD9, CD63 and CD81 exosome detection kits based on time-resolved fluoresence detection were measured using a BMG Labtech microplate reader