Ps capture exosome elisa kit streptavidin hrp

A PS Capture Exosome ELISA Kit (Streptavidin HRP) (FUJIFILM Wako, Japan) was used for semi-quantification of EVs. Cell line-derived EVs or serum were used as the samples. The samples were diluted to the desired ratio using the reaction buffer in the kit. Diluted samples were poured into three wells of the kit (100 μl per well) and incubated overnight with shaking at 4°C and 500 rpm. Subsequent procedures were performed according to the manufacturer’s instructions. Control biotinylated anti-CD63 antibody in the kit or biotinylated anti-aggregated TTR antibody described in the previous section were used for the detection of proteins contained in the EVs. Absorbance spectra were obtained at 25°C using an Enspire Multimode plate reader (PerkinElmer), and the absorbance was measured at 450 nm (reference wavelength: 620 nm). According to the manual, the actual absorbance was obtained by subtracting the blank absorbance from the average absorbance at three points.

Serum levels of EVs expressing either platelet markers (CD41, CD61) or EV marker (CD63) were quantified using the PS Capture Exosome ELISA Kit (Streptavidin HRP) (Wako Pure Chemical Industries, Ltd) as described previously [9 (link)]. Briefly, 100-fold diluted sera (100 μl) were incubated with Tim4-coated plates for 2 h at room temperature (RT). After washing, a biotinylated antibody for each marker protein was incubated for 1 h at RT. Then, horseradish peroxidase (HRP)-labeled streptavidin was incubated for 2 h at RT followed by washing. After incubation with 3,3’,5,5’-tetramethylbenzidine solutions for 30 min, a stop solution was applied. An optical density (OD) was measured at 450 nm as the dominant wavelength and 620 nm as the secondary wavelength. The following antibodies were biotinylated and used for sandwich assays: anti-CD41 mAb (1 μg/mL, clone No. 745201; R&D Systems), anti-CD61 mAb (1 μg/mL, clone No. 256809; R&D Systems), anti-CD63 (component of PS Capture Exosome ELISA Kit; Wako).
CA19-9 was measured using Accuraseed CA19-9 reagents (FUJIFILM Wako Pure Chemical Co., Osaka, Japan).

P‐Exo marker (CD41, CD61, CD9, CD81, and CD63)‐positive EVs in sera were quantified using PS Capture Exosome ELISA Kit (Streptavidin HRP) (Wako Pure Chemical Industries, Ltd) following the manufacture's protocol with modifications. Tim4‐coated plates were incubated with 100 µL of sera (10‐fold dilution: Tim4‐αCD81, 100‐fold dilution: Tim4‐αCD9, Tim4‐αCD63, Tim4‐αCD41, Tim4‐αCD61) for 2 h at room temperature (RT). After washing, biotinylated antibody for each P‐Exo marker was incubated for 1 h at RT. HRP‐labeled streptavidin was then added and incubated for 2 h at RT. After washing, TMB solutions were added. After 30 min, stop solution was applied, and optical density (OD) was measured at 450 nm as the dominant wavelength and 620 nm as the secondary wavelength. The following antibodies were biotinylated and used for sandwich assays: αCD61 mAb (1 µg·mL⁻¹, clone No.: 256809; R&D Systems), αCD41 mAb (1 µg·mL⁻¹, clone No.: 745201; R&D Systems), αCD9 mAb (0.1 µg·mL⁻¹, clone No.: Ts9; Thermo Fisher Scientific K.K.), and αCD81 (0.1 µg·mL⁻¹, SHI‐EXO‐M03; Cosmo Bio Co., Ltd.).