Extracellular Vesicle Quantification Kits

Summary Section 3Introduction 34ExoTESTTM Ready to Use ELISA quantification kit 35Exo-FACS Ready to Use kit 38

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Extracellular Vesicle Quantification Kits

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Exosome research is a relatively novel and exciting field of biomedical research with recent developments of applications in vaccinology and oncologyr. The use of exosomes for assessment of functional state of parent cell in basic and clinical research is currently hampered by the lack of reliable, affordable and optimized tools for quantitative analysis of exosomes in complex biological samples (e.g. plasma or urine). In this section we present ExoTESTTM a technology that allows purification, quantification and characterization of circulating exosomes specific to a cell type or tissue, or to a condition (health vs. disease). Together with ExoTESTTM HBM-LS team has developed a new kit for FACS (Fluorescence Acti-vated Cell Sorting) analysis of exosome markers, Exo-FACS, which allows exo-some FACS profiling without wasting time in exosome isolation by ultracentri-fugation.

1- ExoTESTTM Ready to use kit: ELISA assay for exosome immunocapture, quantification and characterization from human biofluids (plasma, serum, urine, saliva) and cell culture media.

2- Exo-FACS Ready to use kit Ready to use kit which allows exosome isolation from biofluids or cell media and biomarker analysis via FACS.

Products can be purchased directly in our

on-line shop:

www.exotest.eu/online_orders/

Distributors:

www.hansabiomed.eu/index.php/distribu-

tors

IntroductionOrdering information

For information and technical support

contact us at:

info@hansabiomed.eu

Technical support

Catalog 2018Section 3Extracellular Vesicle Quantifi cation Kits

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ExoTESTTM is a patented double sandwich ELISA assay for quantitative and quali-tative analysis of exosomes. In particular, ExoTESTTM is a successful platf orm for exosome quantifi cation and characterization from small amount of human biolo-gical fl uids or cell media and it may be exploited to identify exosomes released by cancer cells in the plasma and urine of tumor patients in various disease conditions. ExoTEST enables reliable and precise quantitative measurement and comparison among samples and individual experiments and provides increased sensitivity in detection of exosomal markers with respect to other analytical methods (i.e FACS, WB).

ExoTESTTM consists of ELISA plates pre-coated with proprietary pan-exosome antibodies enabling specifi c capture of exosomes from diff erent biological samples, including cell culture supernatants and human biological fl uids. Quantifi cation and characterization of exosomal proteins is subsequently performed using appropriate detection antibodies against exosome associated antigens that can be for either generic or cell/tissue-specifi c exosomes. Lyophilized Exosome Standards, characterized for protein content and particle number (NTA) allow the quantifi cation of unknown sample by a standard calibration curve.

Kit components Description

Immunoplate 96 well (12 strips x 8 wells) precoated with specifi c exosome capturing antibody.

Lyophilized Exosome Standards

Exosome Standards from human plasma, serum, urine, saliva or cell medium for calibration curve.

Antibodies for exosome marker detection

Primary anti-human CD9 antibody (HBM proprietary) and sec-ondary antibody HRP conjugated for exosome detection.

Buff ers Sample buff er for antibody dilution and incubation. Washing buf-fer for washing ELISA plate.

Reagents Reagents for signal detection.

• Exosome capture and quantifi cation

from human biofl uids and cell culture

media.

• Comprehensive exosome profi ling.

• Pre-clinical research on non-

invasive biomarkers for detection

and monitoring of a number of

pathological conditions (infl ammation,

cancer, neurodegeneration, etc).

ExoTESTTM: ELISA ready-to-use quantifi cation kit Applications

• Ready to use.

• No initial exosome purifi cation

required.

• User friendly and suitable for multiple

marker analyses.

• Available in TEST format (limited to 3

ELISA strips, 24 wells).

Advantages

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HBM-LS offers different types of ExoTESTTM kit for quantification of Overall Exo-some population from human biofluids (plasma, urine, serum) and from cell cul-ture supernatants. In addition, it is available the ExoTESTTM for Tumor-derived Exosome Enrichment and quantification.

Cat. Code Description Immunoplate

ExoTESTTM for Overall Exosome immunocapture and quantification from human plasma, urine and saliva

HBM-RTK-POF/## Exosome detection performed with anti-human CD9 antibody. Secondary antibody: anti-mouse HRP conjugated.

Transparent

White

ExoTESTTM for Overall Exosome immunocapture and quantification from human serum

HBM-RTK-POS/## Exosome detection performed with anti-human CD9 biotin-conjugated antibody. Signal detected with Streptavidin-HRP conjugated.

Transparent

White

ExoTESTTM for Overall Exosome immunocapture and quantification from cell culture media

HBM-RTK-POC/## Exosome detection performed with anti-human CD9 biotin-conjugated antibody. Signal detected with Streptavidin-HRP conjugated.

Transparent

White

ExoTESTTM for Tumor-derived Exosome immunocapture and quantification from human plasma

HBM-RTK-PTF/## Exosome detection performed with anti-human CD9 antibody. Secondary antibody: anti-mouse HRP conjugated.

Transparent

White

Custom made ExoTESTTM for Specific Exosome immunocapture and quantification

HBM-RTK-CMK HansaBioMed Life Sciences offers the flexibility of creating and designing your own kit by choosing among a wide variety of reagents available in our catalog. For information contact info@hansabiomed.eu

All kits are also available in TEST format, limited to 3 ELISA strips (24 wells). Cat Code: HBM-TRTK-###

Starting material: 100 μl of biological

sample. Whole plasma and serum can

be directly loaded on the plate. It is

recommeded to use concentrate (10X)

urine or cell media prior to the capture

(see page 21).

The detection limit of the assay is lower

than 0.35 μg of overall exosomes which

is the equivalent to less than 50 pg of

targeted exosome protein.

Kit contains Lyophilized Exosome

Standards for assay calibration.

All reagents are shipped and can be stored

at 4°C as described in the datasheet of the

product.

ExoTESTTM: ELISA ready-to-use quantification kitMaterial amount

Detection limit

Assay calibration

Storage conditions

References

Damania, A., Jaiman, D., Teotia, A. K., & Kumar, A. (2018). Mesenchymal stromal cell-derived exo-some-rich fractionated secretome confers a hepatoprotective effect in liver injury. Stem cell research & therapy, 9(1), 31.

Musante, L., Tataruch-Weinert, D., Kerjaschki, D., Henry, M., Meleady, P., & Holthofer, H. (2017). Re-sidual urinary extracellular vesicles in ultracentrifugation supernatants after hydrostatic filtration di-alysis enrichment. Journal of Extracellular Vesicles, 6(1), 1267896.

Zarovni, N., Corrado, A., Guazzi, P., Zocco, D., Lari, E., Radano, G., ... & Chiesi, A. (2015). Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocap-ture approaches. Methods.

Tetta, C., Ghigo, E., Silengo, L., Deregibus, M. C., & Camussi, G. (2013). Extracellular vesicles as an emerging mechanism of cell-to-cell communication. Endocrine, 44(1), 11-19.

Federici, C., Petrucci, F., Caimi, S., Cesolini, A., Logozzi, M., Borghi, M., ... & Fais, S. (2014). Exosome release and low pH belong to a framework of resistance of human melanoma cells to cisplatin.

Logozzi, M., De Milito, A., Lugini, L., Borghi, M., Calabrò, L., Spada, M., ... & Fais, S. (2009). High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients. �(4), e5219.

Catalog 2018Section 3Extracellular Vesicle Quantifi cation Kits

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1. CD9 titration (blue line) of healthy donor plasma exosome standards (HBM-PEP100) and comparison with observed background (red line, only secondary antibody).

2. CD9 exosome marker detection by Western Blott ing on lyophilized exo-somes from human plasma (HBM-PEP100) and recombinant CD9 protein.

Lyophilized Exosomes

RecombinantProtein

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

#1 #2 #3 #4 #5

O.D

. 450

nm

Whole plasma samples

4. Standard curve obtained with Lyophilized Exosome Standards from human plasma healthy donors (HBM-PEP100) with anti-CD9 antibody.

5. CD9 titration of exosomes in 5 diff erent whole plasma from healthy donor.

3. Comparison of ExoTEST analysis and total protein content (BCA test) of diff erent frac-tions of human plasma fractioned by SEC col-umn. EVs are eluted in fractions 6-11, whereas the highest yield of plasma proteins appears from fraction 14.

Plasma sample O.D. 450 nm Exosome μg Particle number in 100 μl of plasma

#1 0,5673 12,869 3,86x10^9

#2 0,6194 14,205 4,26x10^9

#3 0,4425 9,6692 2,90x10^9

#4 0,3100 6,2717 1,88x10^9

#5 0,7853 18,458 5,54x10^9

ExoTESTTM: High sensitivity in detecting low exosome amount

Th e sensitivity of the ExoTEST™ is high-er than western blott ing (Figures 1 and 2). 10 μg of lyophilized exosomes are equivalent to 0.1 ng of recombinant exosomal protein: since the standard curve’s lower concentration is 0.39 μg of lyophilized exosomes (Fig 1), the sensitivity of our test is around 39 pg of protein equivalent.

ExoTESTTM: Exosome binding is highly specifi c

ExoTESTTM presents high specifi city for binding only EVs and not circulat-ing proteins. ExoTESTTM shows positive signal only in fractions containing EVs (Fig 3).

ExoTESTTM: Exosome binding is highly specifi c

y = 0,0349x + 0,0654R² = 0,9979

0

0,4

0,8

1,2

1,6

2

0 10 20 30 40 50 60

O.D

. 450

nm

Exosome µg

Example of exosome quantifi cation performed in 5 unknown plasma samples from healthy donors using the ExoTESTTM (HBM-RTK-POF/TP). Following the binding of Lyophilized Standards and unknown samples onto the ELISA plate, test is run according to the kit protocol and exosome detection is performed with anti-CD9 antibody (HBM-LS). Exosome quantifi cation is fi nally per-formed calculating the quantity of exosomes (expressed in μg) in the 5 un-known samples using the equation ob-tained from the standard curve (Fig 3). Th e particle number contained in 100 μl of plasma is calculated from quantity of exosomes (expressed in μg) accord-ing to the particle concentration (num-ber of particles/ml) indicated in the label of the Lyophilized Exosome Stan-dards (HBM-PEP100, NTA: 3x10^11 particles/ml).

R² = 0,9968

0

0,5

1

1,5

2

0 10 20 30 40 50 60

O.D

. 450

nm

exosome ug

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Exo-FACS allows exosome isolation from biofl uids or cell culture media and FACS analysis of exosome markers. Th e kit consists of EXO-Prep reagent for exosome isolation, 4 µm beads used for the overall capture of pre-isolated exosomes, lyophilized exosomes from cell culture supernatants or human biological fl uids as positive control. Th e characterization of exosomal proteins (membrane-expressed or internal) is subsequently performed using appropriate detection antibodies against exosome associated antigens.

HBM-LS off ers diff erent Exo-FACS kits for staining of exosomal markers from hu-man biofl uids (plasma, urine, serum, saliva) and from cell culture supernatants. Exo-FACS contains reagents for 20 reactions (lyophilized exosomes, beads, an-tibodies and buff ers). Primary antibody included in the kit is against a common exosomal marker (CD9 or CD63) and can be used as a positive control for pro-tein profi ling via FACS analysis.

Cat. Code Description Lyophilized Exosome

StandardDetection antibody

Exo-FACS ready to use kits for analysis of exosome marker from human biofl uids

HBM-FACS-PEP FACS analysis of plasma exosomes

HBM-PEP100 1 vial, 100 μg

Anti human CD9

HBM-FACS-PES FACS analysis of serum exosomes

HBM-PES100 1 vial, 100 μg

Anti human CD9

HBM-FACS-PEU FACS analysis of urine exosomes

HBM-PEP100 1 vial, 100 μg

Anti human CD9

HBM-FACS-PESL FACS analysis of saliva exosomes

HBM-PEP100 1 vial, 100 μg

Anti human CD9

Exo-FACS ready to use kits for analysis of exosome marker from cell culture media

HBM-FACS-C FACS analysis of cell derived exosomes

HBM-###100 *1 vial, 100 μg

Anti human CD63

* Possibility to choose the Exosome Standard from the list of Lyophilized Exosome Standards from cell media available in the section 1 of this catalog, page 5 and 6.

• Exosome isolation and exosome

marker characterization via FACS.

• Comprehensive exosome profi ling.

• Ready to use.

• No initial exosome purifi cation

required.

• Lyophilized Exosome Standards for

positive control included.

• User-friendly and suitable for multiple

marker analyses.

• EXO-Prep for exosome isolation.

• Lyophilized Exosome Standards for

positive control.

• Primary antibody for exosome marker

detection as positive control.

• Secondary antibody Alexa 488.

• Sample buff er, for antibody

incubation.

All reagents are shipped and must be

stored at 4°C.

Exo-FACS: ready-to-use kit for EV FACS analysisApplications

Applications

Kit components

Storage

References

Damania, A., Jaiman, D., Teotia, A. K., & Kumar, A. (2018). Mesenchymal stromal cell-derived exo-some-rich fractionated secretome confers a hepatoprotective eff ect in liver injury. Stem cell research & therapy, 9(1), 31.

Catalog 2018Section 3Extracellular Vesicle Quantifi cation Kits

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Exo-FACS was used for a protein mark-er profi le in exosomes derived from diff erent sources. Exosome binding on FACS-beads was performed by incuba-tion at 4°C overnight. Exosome-bead complex is ready to be labeled with fl u-orophore-conjugated antibodies for specifi c exosome markers. In fi gure 6 is shown a profi le of expression of three diff erent exosome markers in exosomes purifi ed from Melanoma (MM1), Neu-roblastoma (SH) and Glioblastoma (U87) cell supernatants.

6. FACS profi ling of exosomal markers CD9, CD63 and TM9SF4 in purifi ed exosomes from MM1, SH-SY5Y and U87 cell lines.

Exo-FACS: Exosome protein profi ling by Flow Cytometry technique

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