Exosome Antibodies & ELISA Kits

Q
™™
Exosome Antibodies & ELISA Kits
Cat #s
. EXOABxxx, EXOELxxx
User Manual
See page #2 for product storage conditions
(ver. 1-2011-08-16)
A limited-use label license covers this
product. By use of this product, you
accept the terms and conditions outlined
in the Licensing and Warranty Statement
contained in this user manual.
Exosome Antibody and ELISA Kits
Cat.#s EXOAB, EXOEL Series
Contents
List of components
2
Exosome Antibodies
3
I. Recommended protocol
3
II. Serum exosome Western data
4
ExoELISA kit
5
I. ExoELISA principle
5
II. Equipment supplied by user
5
III. Protocol
5
A. Exosome precipitation with ExoQuick/ExoQuick-TC 5
B. Exosome isolated with ultracentrifugation method 6
C. Exosome protein standard
7
D. ELISA procedures
8
E. Sample Data
9
IV. Citations………………………………………………………….9
V. Technical references…………………………………………..9
VI. Technical Support
10
VII. Licensing and Warranty Statement
11
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Page 1
System Biosciences (SBI)
User Manual
List of Components
EXOAB Series
Exosome Antibodies Components
Exosome specific primary antibody (CD63,CD9 ,CD81 or
Hsp70 )
Exosome validated secondary antibody (Goat anti-Rabbit
HRP)
Amount
25 μl
5 μl
The Exosome Antibodies are shipped in blue ice and should be stored at +4°C upon receipt. . Properly stored antibodies are
stable for 6 months from the date received. They can be place at -20°C for long term storage.
EXOEL Series
ExoELISA kit Components
Amount
Exosome binding buffer
20 ml
20X Wash buffer
18 ml
Blocking buffer
30 ml
ExoELISA protein standard*
400 μl
Exosome specific primary antibody (CD63,CD9 or CD81)
2 x 25 μl
Exosome validated secondary antibody (Goat anti-Rabbit HRP)
10 μl
ELISA substrate (Super–sensitive TMB)
6 ml
Stop buffer
6 ml
96 well ExoELISA plate (12x8 well strips)
1 plate
The ExoELISA™ kits are shipped in blue ice and should be stored at +4°C upon receipt. Properly stored kits are stable for 6
months from the date received.
* ExoELISA protein standards should be stored at -20°C upon receipt.
We recommend making single-use aliquots to avoid repeated freezethawing.
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Exosome Antibody and ELISA Kits
Cat.#s EXOAB, EXOEL Series
Exosome Antibodies (Cat# EXOAB series)
I.
Recommended protocol
-for isolating serum exosomes for Western blotting analysis
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
If frozen, thaw 250 µl serum on ice
Centrifuge at 1500 × g for 15 minutes to remove cells and cell debris
Transfer sample supernatant to a centrifuge tube
Combine 250 µl sample + 63 µl ExoQuick™
Mix well by inversion three times
Place at 4ºC for at least 30 minutes
Centrifuge at 1500 × g for 5 minutes
Remove supernatant, keep exosome pellet
Centrifuge at 1500 × g for 5 minutes to remove all traces of fluid (take great care not to disturb the pellet)
1
Add 200 µl RIPA buffer (with appropriate protease inhibitor cocktail added) to exosome pellet and vortex 15 seconds
Place at room temperature for 5 minutes (to allow complete lysis)
Perform standard Bradford protein assay to determine yield.
Add Laemmli buffer2 (with Beta-mercaptoethanol) and heat at 95⁰C for 5 minutes.
Chilled on ice for 5 minutes before loading onto gel
Perform standard SDS-PAGE electrophoresis and Western transfer onto PVDF membrane
Block with 5% dry milk in Tris Buffered Saline + 0.05% Tween (TBS-T) for 1 hour
Incubate blot overnight at 4°C with Exosome specific primary antibody (e.g. CD9) at 1:1000 dilution (5% dry milk in
TBS-T)
Wash 3X with TBS-T
Incubate one hour at room temperature with Exosome validated secondary antibody (Goat-Rabbit-HRP) antibody at
1:20,000 dilution (5% dry milk in TBS-T)
Wash 3X with TBS-T
Incubate blot with chemi-luminescence substrate and visualize on film or other imaging equipment
1
1X RIPA buffer contains:

25mM Tris-HCl pH 7.6

150mM NaCl

1% NP-40

1% sodium deoxycholate

0.1% SDS
2
2X Laemmli buffer contains:

4% SDS

20% glycerol

10% 2-mercaptoethanol

0.004% bromphenol blue

0.125 M Tris-HCl pH 6.8
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System Biosciences (SBI)
II.
User Manual
Serum Exosome Western Data
Western blot analysis of serum exosomes using the recommended protocol. 20 µg of serum exosome proteins were loaded
into each lane. All Exosome specific primary antibody were used at 1:1,000 dilution and Exosome validated secondary
antibody were used at 1:20,000 dilution.
ExoELISA™ - (Cat# EXOEL series)
I.
ExoELISA principle
1.
2.
3.
4.
5.
6.
II.
1.
2.
3.
4.
5.
The ExoELISA kit is designed as a direct Enzyme-Linked ImmunoSorbent Assay (ELISA). The exosome particles and their
proteins are directly immobilized onto the wells of the microtiter plate.
After binding, wells are coated with a block agent to prevent non-specific binding of the detection antibody.
The detection (primary) antibody is added to the wells for binding to specific antigen (e.g. CD63) protein on the exosomes.
A Horseradish Peroxidase enzyme linked secondary antibody (Goat anti-Rabbit) is used for signal amplification and to
increase assay sensitivity.
A colorimetric substrate (Extra-sensitive TMB) is used for the assay read-out. The accumulation of the colored product is
proportional to the specific antigen present in each well.
The results are quantitated by a microtiter plate reader at 450 nm absorbance.
Equipment to be supplied by user
Microtiter plate sealing film/cover
37°C incubator
Microtiter plate shaker
Microtiter plate spectrophotometer with 450 nm absorbance capability
Multichannel pipets (recommended)
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Exosome Antibody and ELISA Kits
III.
Cat.#s EXOAB, EXOEL Series
Protocol
A. Exosome precipitation with ExoQuick/ExoQuick-TC
For simple and quick isolation of exosomes from serum, we recommend using the ExoQuick precipitation solution (Catalog#
EXOQ5A-1 or EXOQ20A-1) and the ExoQuick-TC for isolation of exosomes from tissue culture media and urine samples
(EXOTC10A-1 or EXOTC50A-1).
1.
2.
3.
If frozen, thaw sample on ice
Centrifuge at 3000 × g for 15 minutes to remove cells and cell debris.
Transfer supernatant to a sterile vessel and add the appropriate volume of ExoQuick or ExoQuick-TC.
Incubation time
30 minutes
Overnight
Overnight
Overnight
Overnight
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Bio-fluid
Serum
Ascites fluid
Culture Media
Urine
Spinal fluid
Sample volume
250 l
250 l
5 ml
5 ml
5 ml
Mix well by inversion three times
Place at 4 ºC from 30 minutes to overnight according to table.
Centrifuge at 1500 × g for 30 minutes
Remove supernatant, keep exosome pellet
Centrifuge at 1500 × g for 5 minutes to remove all traces of fluid (take great care not to disturb the pellet)
Add 200 µl Exosome Binding buffer to exosome pellet and vortex 15 seconds
Incubate at 37 ºC temperature for 20 minutes
Centrifuge at 1500 × g for 5 minutes to remove all residual precipitation solution
Transfer supernatant to new centrifuge tube on ice
Exosome protein is now ready for immobilization onto micro-titer plate
Proceed to section - C. Exosome protein standard curve
B. Exosome isolated with ultracentrifugation method
1.
2.
3.
4.
5.
ExoQuick volume
63 l
63 l
-
Performed exosome isolation by the user’s preferred ultra-centrifugation protocol
Carefully remove all traces of supernatant, keep exosome pellet
Resuspend exosome pellet in 200 µl of Exosome binding buffer and vortex 15 seconds
Incubate at room temperature for 10 minutes and keep on ice.
Exosome protein is now ready for immobilization onto micro-titer plate
Proceed to section - C. Exosome protein standard curve
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ExoQuick-TC voulme
1 ml
1 ml
1 ml
System Biosciences (SBI)
User Manual
C. Exosome protein standard curve
A standard curve should be prepared each time the assay is performed
1. Thaw ExoELISA protein standard on ice
2. Dilute ExoELISA protein standard by performing serial dilutions with Exosome Binding buffer in microcentrifuge tubes
3. Suggested dilutions for standard curve:
Tube
# Exosomes
Dilution factor
ExoELISA protein standard
0
1
2
3
4
5
6
7
0
1.35x1010
6.75x109
3.37x109
1.68x109
8.44x108
4.21x108
2.10x108
Blank
1
1:2
1:4
1:8
1:16
1:32
1:64
60 l
60 l
60 l
60 l
60 l
60 l
60 l
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Exosome binding
buffer
60
60 l
60 l
60 l
60 l
60 l
60 l
Exosome Antibody and ELISA Kits
Cat.#s EXOAB, EXOEL Series
D. ELISA procedures
Before starting
1. The ExoELISA micro-titer plate is provided in a convenient 12 well X 8 strip format. We recommend using at least one strip
for the standard curve and additional strips depending on the number of samples tested. Unused 8-well strips can be
removed and stored at room temperature for later use.
2.
Dilute stock 20X Washing buffer into 1X working Wash buffer with purified water (each 8-well strip requires approximately
10 ml of 1X Washing solution)
ELISA assay
1. Add 50 µl of prepared protein standards and exosome protein sample to the appropriate well of the micro-titer plate
2. Cover plate with sealing film/cover
3. Incubate the plate at 37ºC from 2 hours to overnight (recommended)
4. After incubation step, invert the plate to empty all contents.
5. Wash the plate 3 times for 5 minutes each with 100 µl 1X Wash buffer
- A micro-titer plate shaker is recommend for all subsequent the washing and incubation steps
- Residual liquid should be removed by hard-tapping the plate on fresh paper towels, while taking care not to let the wells dry
out completely
6. Dilute Exosome specific primary antibody (CD63, CD9 or CD81) 1:100 in 1X blocking buffer and add 50 µl of to each well
and incubate at room temperature for 1 hour with shaking
7. Wash the plate 3 times for 5 minutes each with 100 µl 1X Wash buffer
8. Dilute Exosome validated secondary antibody 1:5,000 1X blocking buffer and add 50 µl to each well and incubate at room
temperature for 1 hour with shaking
9. Wash the plate 3 times for 5 minutes each with 100 µl 1X Wash buffer
10. Add 50 µl of Super-sensitive TMB ELISA substrate and incubate at room temperature for 15 to 45 minutes with shaking
-15 to 45 minutes substrate incubation time is optimized for the recommended exosome protein standard curve
- Further optimization maybe required by the user for individual sample.
11. Add 50 µl of Stop buffer to provide a fixed endpoint for the assay
- Note that the initial color of a positive sample is blue and the color changes to yellow when Stop buffer is added
12. Quantitate results with a spectrophotometric plate reader at 450 nm absorbance
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System Biosciences (SBI)
User Manual
E. Sample Data
Page 8
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Exosome Antibody and ELISA Kits
IV.
Cat.#s EXOAB, EXOEL Series
Citations
As featured in: Exosome Isolation for Proteomic Analyses and RNA Profiling Douglas D. Taylor, Wolfgang Zacharias and
Cicek Gercel-Taylor, Serum/Plasma Proteomics, Methods in Molecular Biology, 2011, Volume 728, Part 4, 235-246, (PDF) »
Tae Hoon Lee, Esterina D'Asti, Nathalie Magnus, Khalid Al-Nedawi, Brian Meehan and Janusz Rak. Review: Microvesicles as
mediators of intercellular communication in cancer—the emerging science of cellular 'debris'. Seminars in Immunopathology DOI:
10.1007/s00281-011-0250-3. (PDF) »
V.
Technical References
Adachi T, Nakanishi M, Otsuka Y, Nishimura K, Hirokawa G, Goto Y, Nonogi H, Iwai N. Plasma microRNA 499 as a biomarker of
acute myocardial infarction. Clin Chem. 2010 Jul;56(7):1183-5.
De Smaele E, Ferretti E, Gulino A. MicroRNAs as biomarkers for CNS cancer and other disorders. Brain Res. 2010 Jun
18;1338:100-11.
Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O'Briant KC, Allen
A, Lin DW, Urban N, Drescher CW, Knudsen BS, Stirewalt DL, Gentleman R, Vessella RL, Nelson PS, Martin DB, Tewari M.
Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A. 2008 Jul
29;105(30):10513-8.
Laterza OF, Lim L, Garrett-Engele PW, Vlasakova K, Muniappa N, Tanaka WK, Johnson JM, Sina JF, Fare TL, Sistare FD, Glaab
WE. Plasma MicroRNAs as sensitive and specific biomarkers of tissue injury. Clin Chem. 2009 Nov;55(11):1977-83.
Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a
novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007 Jun;9(6):654-9.
Pegtel DM, Cosmopoulos K, Thorley-Lawson DA, van Eijndhoven MA, Hopmans ES, Lindenberg JL, de Gruijl TD, Wordinger T,
Middeldorp JM. Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci USA; 2010 Apr 6; 107(14):6328-33.
Mathivanan, S. and Simpson, R.J. ExoCarta: A compendium of exosomal proteins and RNA. Proteomics. 2009.21, 4997-5000.
Thery C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 2009. 8, 581-93.
Michael A, Bajracharya SD, Yuen PS, Zhou H, Star RA, Illei GG, Alevizos I. Exosomes from human saliva as a source of
microRNA biomarkers. Oral Dis; 2010 Jan; 16(1):34-8.
Luo SS, Ishibashi O, Ishikawa G, Ishikawa T, Katayama A, Mishima T, Takizawa T, Shigihara T, Goto T, Izumi A, Ohkuchi A,
Matsubara S, Takeshita T, Takizawa T. Human villous trophoblasts express and secrete placenta-specific microRNAs into
maternal circulation via exosomes. Biol Reprod; 2009 Oct; 81(4):717-29.
Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.
Gynecol Oncol; 2008 Jul; 110(1):13-21.
Simpson RJ, Lim JW, Moritz RL, Mathivanan S. Exosomes: proteomic insights and diagnostic potential. Expert Rev Proteomics.
2009 Jun;6(3):267-83. Review.
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VI.
User Manual
Technical Support
For more information about SBI products and to download manuals in PDF format, please visit our web site:
http://www.systembio.com
For additional information or technical assistance, please call or email us at:
System Biosciences (SBI)
265 North Whisman Road.
Mountain View, CA 94043
Phone: (650) 968-2200
(888) 266-5066 (Toll Free)
Fax:
(650) 968-2277
E-mail:
General Information: info@systembio.com
Technical Support: tech@systembio.com
Ordering Information: orders@systembio.com
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Exosome Antibody and ELISA Kits
Cat.#s EXOAB, EXOEL Series
VII. Licensing and Warranty Statement
Limited Use License
Use of the ExoAB antibodies and ExoELISA Kits (i.e., the “Product”) is subject to the following terms and conditions. If the terms and conditions are not acceptable,
return all components of the Product to System Biosciences (SBI) within 7 calendar days. Purchase and use of any part of the Product constitutes acceptance of the
above terms.
The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions:
The Product shall be used by the purchaser for internal research purposes only. The Product is expressly not designed, intended, or warranted for use in humans
or for therapeutic or diagnostic use.
The Product may not be resold, modified for resale, or used to manufacture commercial products without prior written consent of SBI.
This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research.
SBI has pending patent applications related to the Product. For information concerning licenses for commercial use, contact SBI.
Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement. Use of the Product for
any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned. SBI disclaims any and all responsibility for
injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein.
Limited Warranty
SBI warrants that the Product meets the specifications described in this manual. If it is proven to the satisfaction of SBI that the Product fails to meet these
specifications, SBI will replace the Product or provide the purchaser with a refund. This limited warranty shall not extend to anyone other than the original purchaser of
the Product. Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product.
SBI’s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price. SBI’s liability does not extend to any damages arising from
use or improper use of the Product, or losses associated with the use of additional materials or reagents. This limited warranty is the sole and exclusive warranty. SBI
does not provide any other warranties of any kind, expressed or implied, including the merchantability or fitness of the Product for a particular purpose.
SBI is committed to providing our customers with high-quality products. If you should have any questions or concerns about any SBI products, please contact us at
(888) 266-5066.
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