Q: Do I have to prepare a standard for every assay? And do I have to use Wako MagCapture Exosome Isolation Kit PS for preparation of the standard?

A: When you perform a quantitative assay, prepare an extracellular vesicle sample as standard. Although an extracellular vesicle sample purified by ultracentrifugation or polymer precipitation may also be used as standard, an extracellular vesicle sample purified by the PS affinity method based on the principle identical with that of the assay is recommended (see the instruction manual included in the kit for details of the preparation method).

Q: Why does this kit include no standard?

A: The standard and assay samples must be derived from identical cell species, because the type and amounts of surface marker proteins on extracellular vesicles secreted may vary depending on the cell type. Therefore, this kit does not include a standard. Prepare a standard purified from culture supernatant of cells identical with the source cells of assay samples.

Q: Is this kit compatible with direct assay of serum and plasma samples?

A: This kit is not recommended for direct assay of serum and plasma samples from human, mouse, and rat, because the secondary antibody for detection included in it reacts with human, mouse, and rat IgG non-specifically. However, this kit is compatible with qualitative assay of extracellular vesicle samples purified from serum and plasma specimens using MagCapture Exosome Isolation Kit PS. It is also compatible with qualitative assay of extracellular vesicles purified by ultracentrifugation or polymer precipitation.

Q: Is this kit compatible with direct assay of cell culture supernatant samples?

A: This kit is compatible with direct assay of cell culture supernatant from both serum-free and FBS-supplemented media because primary antibody (Anti-CD63 antibody) and secondary antibody included in the kit don’t react with bovine IgG non-specifically. Utilize this kit for both quantitative and qualitative analyses of extracellular vesicles in cell culture supernatant samples.

Q: Can I replace the primary antibody with another antibody?

A: Yes. Choose a mouse antibody against the surface marker you want to detect and investigate the optimal concentration according to the instruction manual.

Q: How can I store the remaining reagents?

A: See [6. The storage method of each reagent when the kit is separately used] in the instruction manual included in the kit.

Q: Is the detection sensitivity of this kit higher than other methods?

A: This kit has been confirmed to detect extrcellular vesicles at a sensitivity higher than those of ELISA methods involving antibody immobilization or direct immobilization of purified samples on the plate. In addition, a correclation between ELISA results obtained with this kit and Western Blotting results has been confirmed.

Q: How long is the operation time for ELISA using this kit?

A: The entire kit process takes approximately 5 hours, including immobilization of diluted cell culture supernatant specimens or purified and diluted extracellular vesicle samples onto a 96-well plate for 2 hours, reaction with the primary antibody for 1 hour, reaction with the secondary antibody for 1 hour, and reaction with tetramethylbenzidine (TMB) for 30 minutes. With washing and other operations included, the assay is completed in approximately 5 hours. After addition of Stop Solution, measure the absorbance at the main wavelength 450 nm and the complementary wavelength 620 nm (600 - 650 nm).

Q: Is an Exosome Capture 96 Well Plate compatible with recycling?

A: No. An Exosome Capture 96 Well Plate is not compatible with recycling by regeneration, because Stop Solution denatures proteins on the plate.

Q: Is there any step that can be carried over to next day?

A: Immobilization of individual samples onto the plate may be prolonged up to overnight at 4°C without any problem.

Q: How much is the minimum sample amount required for detection using this kit?

A: Extracellular vesicles corresponding to 1 ng protein are detectable using this kit. The detection limit of extracellular vesicles purified from COLO201 cell culture supernatant was 11 pg (it has been confirmed that the detection limit varies depending on the cell strain).

Q: How much sample volume is required for direct assay of culture supernatant?

A: Culture supernatant of a few µL in volume (1-5 µL) is sufficient for assay. This is recommended for monitoring of change in number of extracellular vesicles in culture medium over time and assay of new cell culture supernatant. However, the number of extracellular vesicles in culture medium may be limited depending on the cell species (e.g., iPS cells) and the sample volume per well used for ELISA should be investigated as necessary. If the number of extracellular vesicles in the sample is unknown, use of undiluted culture supernatant samples (100 µL) is recommended.

Q: Are there any primary antibodies recommended for detection?

A: The following antibodies have been confirmed to be compatible with ELISA at our Laboratories.

Antigen	Reactivity	Antibody	Manufacturer	Use
CD63	Human	Mouse anti-CD63 monoclonal antibody (3-13)	Wako, Catalog ID: 012-27063	WB, ELISA
CD81	Human	Mouse anti-CD81 monoclonal antibody (M38)	Novus, Code: NBP1-44861 (Wako 550-30161)	ELISA
CD9	Mouse	Rat anti-CD9 monoclonal antibody (MZ3)	Bio Legend, Code: 124802	ELISA
CD63	Mouse	Rat anti-CD63 monoclonal antibody (NVG-2)	Bio Legend, Code: 143902	ELISA
CD81	Mouse	Armenian hamster anti-CD81 monoclonal antibody (Eat-2)	Bio Legend, Code: 104902	ELISA

Q: My detection results are not satisfactory. Could you tell me what to check for?

A: Check if any of the reagents has been expired. When you fail to detect a positive signal even with Control Primary Antibody Anti CD63 (100 × ) (included in this kit), it may be ascribable to an expression level of CD63 under the detection limit or some other cause. Please inquire to us in such a case.