Materials Not Included in the Kit

• Microtiter plate sealing film/cover
• Micropipettes (10 to 1000 μL)
• Multichannel micropipette (recommended)
• Multichannel micropipette reservoir
• Microtiter plate shaker
• Microplate reader (capable of measuring at a wavelength of 450nm)
• Plate washer

Before starting

• Fresh dilute CD63 Probe (2500X) to 2500-folds in Probe Dilution Buffer before step # 6.e.g. For 1 plate, add 4 μL of CD63 Probe (2500X) into 10 mL of Assay Buffer. Mix by inverting the tube.
• Fresh dilute HRP-conjugated Secondary Probe (2500X) to 2500-folds in Probe Dilution Buffer before step # 8. e.g. For 1 plate, add 4 μL of HRP-conjugated Secondary Probe (2500X) into 10 mL of Assay Buffer. Mix by inverting the tube.

 ELASA assay

• Add 100 µl of freshly prepared Standard Dilutions (see protocol above) and exosome samples to the appropriate well of the 96 Well Plate.
• Seal the plate with plate seal and incubate it at 4°C overnight.
• Remove the plate seal, discard all the reaction solution. Wash each well with 350 µL of 1X Wash buffer, and then discard the buffer carefully. Repeat this step for 3 times. After the final wash, empty the wells and firmly tap the plate on a lint free paper towel to remove any remaining wash buffer. *  Note: Do not use suction/aspirate to directly suck the bottom of the well.
• Add 300 μL of Blocking Buffer to each well. Seal the plate with plate seal and incubate it at room temperature for 1 h on the Microtiter plate shaker (100 rpm).
• Remove the plate seal, discard all the reaction solution. Wash each well with 350 µL of 1X Wash buffer, and then discard the buffer carefully. Repeat this step for 3 times. After the final wash, empty the wells and firmly tap the plate on a lint free paper towel to remove any remaining wash buffer. *  Note:  Do not use suction/aspirate to directly suck the bottom of the well.
• Add 100 µL of dilute CD63 Probe (1X) to each well. Seal the plate with plate seal and incubate it at room temperature for 1 h on the Microtiter plate shaker (100 rpm).
• Remove the plate seal, discard all the reaction solution. Wash each well with 350 µL of 1X Wash buffer, and then discard the buffer carefully. Repeat this step for 3 times. After the final wash, empty the wells and firmly tap the plate on a lint free paper towel to remove any remaining wash buffer. *  Note:   Do not use suction/aspirate to directly suck the bottom of the well.
• Add 100 µL of dilute HRP-conjugated Secondary Probe (1X) to each well. Seal the plate with plate seal and incubate it at room temperature for 1 h on the Microtiter plate shaker (100 rpm).
• Warm the Substrate Buffer to room temperature.
• Remove the plate seal, discard all the reaction solution. Wash each well with 350 µL of 1X Wash buffer, and then discard the buffer carefully. Repeat this step for 3 times. After the final wash, empty the wells and firmly tap the plate on a lint free paper towel to remove any remaining washbuffer. *  Note:  Do not use suction/aspirate to directly suck the bottom of the well.

• Add 100 μL of Substrate Buffer to each well, and then incubate the plate in dark at room temperature for 15 minutes. Add 100 μL of Stop Buffer to each well and read immediately. *  Note:  The initial color of a positive sample is blue and the color changes to yellow when Stop Buffer is added.

• Measure the absorbance with a microplate reader at 450 nm.
• Plot the standard solutions data as mean OD 450.
• Calculate each sample concentration. *  Note: The ideal incubation time varies depending on laboratory conditions and/or the instrument utilized. It's highly recommended to run a sample set of standards beforehand to fine-tune the assay before analyzing sensitive samples. This preliminary step assists in identifying the optimal experimental conditions for your study.

In this Indirect ELASA assay, exosomes are captured intact on the high protein binding microtiter plate. Then, CD63 probe is added, which binds to CD63 on the captured exosomes. Following wash steps, HRP-conjugated Secondary Probe and TMB solution is added, leading to a color change proportional to the amount of exosomes. The colorimetric signal is quantified using a microplate reader, providing a sensitive and specific method for exosome quantification.