We developed PS Capture Exosome ELISA Kit by applying affinity binding of Tim4 protein with exosomes. This kit is capable of detecting exosomes at a sensitivity higher than that of conventional ELISA methods immobilization of antibodies against exosome surface markers. Exosomes in samples such as culture supernatants and serum are captured by Tim4 protein on adry plate in the presence of calcium ion. The captured exosomes are detected by a primary antibody against an exosome surface marker protein and a labeled secondary antibody. While a mouse anti-CD63 monoclonal antibody is provided with the kit, a user-provided mouse primary antibody against any other exosome surface marker may also be used for exosome detection.

The greatest feature of this kit is that it provides exosomes detection with higher sensitivity than that of Western Blot analysis and conventional product for exosome ELISA. First, the detection limit for exosomes in Western Blotting was examined for comparison with this kit (Figures 1a and b). Western Blot analysis of exosomes purified from COLO201 cells (of human colon adenocarcinoma origin) with an anti-CD63 monoclonal antibody detected exosomes in an amount as small as 75 ng on the protein basis. Next, the detection limits of this kit for exosomes purified from K562 cells (of human leukemia origin) and COLO201 cells were determined to be 49.9 pg and 10.9 pg, respectively, demonstrating that this kit had a detection sensitivity more than 1,000 times higher than that of Western Blotting (Figure 1c). Considering that the detection limits of conventional products for exosome ELISA range approximately several ng to several μg (refer to instruction manuals for individual products), the present results demonstrated that this kit utilizing affinity binding of exosomes to Tim4 via PS has a sensitivity more than 100 times higher than those of conventional ELISA methods involving immobilization of an antibody against an exosome surface protein marker.

PS ELISA detected the marker proteins with 50-1,000 times higher sensitivity than WB.

 

Fig. 1 Comparing the detection sensitivities of Western Blot and PS ELISA

(a), (b) Result of sensitivity by Western Blot with each of anti-CD63 antibody (supplier A and Wako: Code No. 012-27063).
Sample： purified extracellular vesicles from cell culture supernatant of COLO201 cells with MagCaptureTM Exosome Isolation Kit PS (Code No. 293-77601)

★：detection limit by Western Blot

(c) Result of limit by PS ELISA

A standard curve was prepared using blank value of buffer and absorbance value of 2-fold serial dilution samples of extracellular vesicles purified from cell culture supernatant of K562 cells and that of COLO201 cells with MagCapture Exosome Isolation Kit PS. Then, the detection limit of purified extracellular vesicles of K562 cells and COLO201 cells were calculated using its standard curve. (each dilution point: n=6, blank: n=12)