• Sensitive qualitative analysis (50 to 1000 folds more sensitive than WB in detection)
• Direct relative quantitation of extracellular vesicles in conditioned medium and body fluid specimens
• Application to detection systems using antibodies from various animal species and lectins
• Microanalysis with small amounts of extracellular vesicles
  (only 1/10 to 1/1000 of the sample amount for WB)

Exosome Capture 96 Well Plate which is pre-coated with protein that specifically binds to phosphatidylserine (PS) on the surface of extracellullar vesicles (EVs) capture EVs with Ca²⁺, Then biotinylated antibody for the surface marker protein of EVs is used as a primary detection and HRP-conjugated streptavidin is used as a secondary detection.

Dilution linearity of four specimens, ① human serum diluent ② human heparin plasma diluent ③ human EDTA plasma diluent (2 specimens, 4-step serial dilution each) and ④ COLO201 cells cell culture supernatant diluent (4-step serial dilution), was assessed by measuring the concentration of exosomes (CD63 detection) with standard curve obtained using extracellular vesicles purified from cell culture supernatant of COLO201 cells.

①Serum

②Heparin plasma

③EDTA plasma

④COLO201 cell culture supernatant

Samples in serum and heparinized plasma have given favorable linearity when diluted 2 folds or more.
Even samples in EDTA-treated plasma will give favorable linearity when diluted 5 folds or more.

Microplate wells, pre-coated with anti-CD antibodies (CD9, CD63, CD81) and PS Affinity (Tim4), were incubated with pretreated ※ 1 cell culture supernatants of iPSC, COLO201 and HEK293T cells. Captured exosomes on the plate were detected using biotinylated anti-CD antibodies (CD9, CD63, CD81).

※ 1 Pretreatment condition: 10,000 x g, 30min.

Almost all of the exosomes derived from various cell lines were efficiently captured by Tim4 compared with anti-CD9, 63, 81 antibodies.

Pretreated^※2 pooled normal human serum, EDTA-treated plasma, and heparinized plasma were spiked with exosomes isolated and purified from COLO201 cell conditioned medium using MagCapture Exosome Isolation Kit PS (Code No. 293-77601) at various concentrations. The spiked exosomes in a sample were detected using biotin-labeled anti-EpCAM^※3 antibody (MBL), and the recovery rate of exosomes from the sample was calculated based on the standard curve prepared using exosomes purified from COLO201 cell conditioned medium.

※ 2 Pretreatment condition: 10,000 x g, 30min.　　※ 3 EpCAM: Epithelial cell adhesion molecule

The spiked recovery rates based on the detection of EpCAM fell within a range of 100%±10%, showing favorable recovery performance.

Extracellular vesicles in untreated FBS, commercial EV-depleted FBS and ultracentrifugation (UC)-treated FBS ^{※ 4} were detected using Biotinylated anti-CD9 antibody ^{※ 5}.

Capture: Tim4 / Detection: Biotinylated anti-CD9 antibody

※ 4 Ultracentrifugation condition: 160,000 x g, 16h
※ 5 Anti-CD9 antibody (Code No. 014-27763) was biotinylated using Biotin Labeling Kit-SH (Code No. 348-90941).

PS Affinity ELISA System enables test of residual extracellular vesicles in EV-depleted FBS.

Exosomes in five-fold dilution of pretreated^※6 cell culture supernatants of iPS cells were detected using Biotinylated anti-CD63 antibody (Code No. 019-27713) and Biotinylated rBC2LCN lectin^※7.

Capture: Tim4 / Detection: Biotin labelled compounds

※ 6 Pretreatment condition: 10,000 x g, 30min.
※ 7 rBC2LCN binds specifically to Fucα1-2Galβ 1-3GalNAc (GlcNAc) and is known as a undifferentiated marker of iPS and ES cells. rBC2LCN (Code No. 029-18061) was biotinylated using EZ-Link™ Sulfo-NHS-LC-LC-Biotin (ThermoFisher).

Reference: S. Saito, et al., Sci. Rep., 8, 3997 (2018)

PS Affinity ELISA is applicable for sugar chain analysis on exosomes with a various combination of lectins.

From 1 mL of pretreated ^{※ 8} COLO201 cell conditioned medium, exosomes were isolated and purified by PS affinity method or polymer precipitation method (competiter A) and then measured for signals of CD63, an exosome marker, using PS Capture Exosome ELISA Kit (streptavidin HRP). From absorbance of each sample, the amount of residual exosomes in a post-purification sample and the recovery rate of exosomes were determined for comparison.

※ 8 Pretreatment condition: 10,000 x g, 30min.

Calculation of recovery efficiency

Input: Measured value of 100-fold diluted conditioned medium^※Ⅰ
Remaining sEVs: Measured value of post-purification 100-fold diluted conditioned medium ^※Ⅰ
Recovered sEVs: Measured value of 1000-fold diluted purified sample ^※Ⅱ

※Ⅰ Measured value of 100-fold diluted sample has been confirmed to fall within the measurement range. In the preliminary investigation, a dilution ratio appropriate for ELISA is recommended to be calculated for each sample
※Ⅱ Exosome samples purified with this kit and polymer reagents are theoretically concentrated 10 folds. (e.g. 1 mL of input → 100μL of purified exosomes) Value obtained by multiplying the concentration ratio by the dilution ratio for ELISA corresponds to integrated dilution ratio of the purified exosome for ELISA.

The ELISA system allowing highly sensitive detection of exosom es will ease efficiency comparison among exosome recovery methods and validation of the method.