CD9 / CD63 ELISA kit (for quantification of human-derived exosomes)

Direct quantification of exosomes from blood samples and cell culture supernatants!

Background

Exosomes are vesicles with a diameter of 30-200 nm that are secreted from almost all cells that make up the body, and are present in all body fluids such as blood and urine ^(1)-(5) . It is also secreted into the culture supernatant of animal cells in vitro . Like cells, exosomes are surrounded by a lipid bilayer membrane, with membrane proteins on the surface and proteins and microRNAs inside. Exosomes are thought to be responsible for intercellular communication through the functions of these proteins and microRNAs in target cells that have taken up exosomes ⁽⁶⁾ . One of the structural features of exosomes is the tetraspanin family present on their surface. CD9 and CD63 are member molecules and surface markers of exosomes ⁽⁷⁾ .

Exosome quantification methods include substituting the amount of protein encapsulated in exosomes and particle analysis by nanotracking ⁽⁸⁾ , but these methods require exosomes to be purified once by ultracentrifugation. Methods for directly quantifying exosomes in body fluids and cell culture supernatants are extremely limited, and no general method has been developed so far.

This product can relatively quantify exosomes with both CD9 and CD63 molecules on their surface contained in body fluids and cell culture supernatants by sandwich ELISA using high-performance antibodies against exosome markers CD9 and CD63. 

Features

• Human-derived exosomes contained in blood samples and cell culture supernatants can be directly quantified
• Sensitivity: 3.125 pg/mL, measurement range: 3.125-200 pg/mL
• No special equipment required, can be measured with a normal plate reader (wavelength: 450 nm)
• Stability and reproducibility are ensured by using a CD9/CD63 fusion protein (standard protein) instead of exosomes themselves, which lack storage stability, as a standard reagent.
• Compensation with CD9/CD63 fusion protein (standard protein) enables relative quantification of each sample
• Capture exosomes with immobilized CD9 antibody (12A12) and detect with HRP-labeled CD63 antibody (8A12)

Composition

• Anti-CD9 antibody immobilized 96-well plate
• Standard protein (2,000 pg/mL)
• Assay buffer
• Wash buffer (10X)
• HRP-conjugated anti-CD63 antibody (500X)
• Substrate solution
• Stop solution _( 2N H2SO4 )
• plate seal

Product data

Overview of standardization and relative quantification with standard proteins

A standard curve was drawn using the CD9/CD63 fusion protein as the standard protein (Figure 1.A), for example, 5 pg of CD9/CD63 fusion protein = 1 unit, the measured OD _{450 is approximately 0.7. Figure 1.B, which plots the OD 450} readings of exosomes from MDA-MB-231 cells , a reading of 0.7 corresponds to approximately 50 ng, which are considered CD9-positive and CD63-positive exosomes. can do. In this way, it is possible to directly compare the amount of exosomes in samples between different samples, or even between different experiments, by normalizing the standard curve using the CD9/CD63 fusion protein for each assay.

 

 

 

 

Figure 1. Overview of normalization and relative quantification with CD9/CD63 fusion proteins.

Human Serum Dilution Linearity Test and Spike Recovery Test

Human serum was added to the CD9/CD63 fusion protein (standard protein) and exosomes (collected by ultracentrifugation) to create a dilution series to examine whether linearity could be ensured. Measured by adding 25 µL of serum to a 100 µL ELISA reaction, the dilution linearity is maintained, similar to the buffer-based standard curve.

 

Figure 2. Dilution linearity test
Amount of serum added per well: 25 µL
A: Amount of CD9/CD63 fusion protein (standard protein) added: 20 pg B: Amount of exosomes derived from MDA-MB-231 cells added: 100ng
CD9/CD63 When human serum was added to the fusion protein (standard protein) and MDA-MB-231 cell-derived exosomes and serially diluted 2-fold, linearity similar to the buffer-based standard curve was shown.

 

 Next, when CD9/CD63 fusion protein (standard protein) or exosomes (collected by ultracentrifugation) were added in the presence of human serum, we examined the probability that the added amount could be recovered. The CD9/CD63 fusion protein (standard protein) showed a recovery rate of around 100% even in the presence of 20 µL/well of serum. Got it. Combined with the results of the dilution linearity test, it is considered that the effect of serum components in this assay system is small when the amount of added serum is up to 25 µL.

Figure 3. Spike and recovery test
Amount of serum added per well: 1, 2.5, 5, 10, 25 µL A: Amount of CD9/CD63 fusion protein (standard protein) added per well: 2, 8, 16 pg,
B: Well MDA-MB-231 cell-derived exosomes added per well: 8, 32, 80 ng
CD9/CD63 fusion protein (standard protein) yields around 100% recovery even in the presence of 25 µL of serum per well.
Although there were variations in MDA-MB-231 cell-derived exosomes, the results were generally similar to those of the CD9/CD63 fusion protein (standard protein).

[Product data]　Addition recovery test measured value

Measurement of cell culture supernatant samples

Various cell lines (HCT116, HT29, AsPC-1, MDA-MB-231, PC3) were cultured for 8 days in medium containing 10% fetal bovine serum (FBS), and the centrifugation supernatant was sampled. It was diluted appropriately so that the measured value fell within the range of the standard curve, and measured together with the diluted CD9/CD63 fusion protein (standard protein). The assay provides relative quantification against a CD9/CD63 fusion protein (standard protein). Based on the measurement results of the CD9/CD63 fusion protein (standard protein), draw a standard curve with the standard protein amount on the horizontal axis and the absorbance on the vertical axis (Fig. 4.A). By comparing this standard curve with the absorbance of the sample, the amount of exosomes in the sample can be calculated as the amount equivalent to the standard protein (Fig. 4.B). By drawing a standard curve for each experiment, exosome abundance between different experiments can be directly compared.

Figure 4. Measurement of cell culture supernatant sample
Cancer cell culture supernatant (cultivated at a density of 1x10 ⁴ cells/cm ² for 8 days) was serially diluted 2-fold and measured at N = 2.
A: Standard curve for CD9/CD63 fusion protein
B: Measurement example of cell culture supernatant

References

1. J. Skog, T. Wurdinger, S. van Rijn, DH Meijer, L. Gainche, M. Sena-Esteves, WT Jr. Curry, BS Carter, AM Krichevsky and XO Breakefield: Nat Cell Biol. , 10 , 1470 (2008 ).
2. T. Pisitkun, RF Shen and MA Knepper: Proc Natl Acad Sci USA ., 101 , 13368 (2004).
3. S. Runz, S. Keller, C. Rupp, A. Stoeck, Y. Issa, D. Koensgen, A. Mustea, J. Sehouli, G. Kristiansen and P. Altevogt: Gynecol Oncol ., 107 , 563 (2007) .
4. S. Keller, AK Konig, F. Marme, S. Runz, S. Wolterink, D. Koensgen, A. Mustea, J. Sehouli and P. Altevogt: Cancer Lett ., 278 , 73 (2009).
5. C. Lasser, V. Seyed AlikhS. Gabrielsson, J. Lotvall and H. Valadi: J Transl Med ., 9 , 9 (2011).
6. Y. Naito, Y. Yoshioka, Y. Yamamoto and T. Ochiya: Cell Mol Life Sci ., 74 , 697 (2017).
7. A. Zoraida and M. Yáñez-Mó: Front Immunol ., 5 , 442 (2014).
8. V. Filipe, A. Hawe and W. Jiskoot: Pharm Res ., 27 , 796 (2010).

 

Catalog Number	Product Name	Size
CSR-EXH0102EL	CD9/CD63 Exosome ELISA Kit	1 KIT [96 tests]