Size Control Single Tope

EVix Single-Tope Kit 

Research Use Only  Not for Diagnostic Use

Description: The EVix Single-Tope ELISA kit brings revolutionary screening capabilities to EV assays.  Antigen specific EVs are captured using the Multi-Tope capture plates.  Unlike the Multi-Tope assay, detection of the captured EVs is carried out a selected biotinylated antibody of choice.   The detection antibody can be selected from over 115 candidates available from Ancell, or can be sourced from other suppliers.  Antigen positive populations identified using the Multi Tope assay, can now be further probed.  Identification of multiple antigen positive populations leads to further characterization of an EV preparation.

The Single-Tope ELISA kit contains duplicate wells for each capture antibody, one is used to detect the antigen specific captured EVs and the other is used as a negative control for the biotinylated detection antibody.  The signals generated are specific for EVs captured within the individual wells.

This assay kit allows assessment of different multiple antigens in an EV sample.  For example do EPCAM positive EVs express the same antigens as MHC II positive EVs?  Selecting both EPCAM and MHCII as detection antibodies and running them against the entire panel of capture antibodies, one is able to ascertain the pattern of expression of associated antigens for both EPCAM and MHCII positive EVs .  Depending upon sample source, the distribution of the antigens may vary significantly.   Changes in EV populations from a particular sample can also be tracked by the extensive antigen expression profiling.  Linking EV antigen expression profiles to particular physiological conditions can be easily investigated.

The Single-Tope ELISA kit comes complete with all reagents needed to run the 256 well assay except for a biotinylated detection antibody.  The detection antibody is provided by the user.  Biotinylated antibody from Ancell can be ordered and used at 2-4 ug/ml concentrations diluted in wash buffer.  Alternatively other biotinylated antibody can be used.  To assure maximum signal, an overnight capture is followed by a 1 hr detection incubation.  With minimal effort, an EV preparation can be finger printed for the presence of 115 unique antigens associated with expression of the researcher selected antigen of choice.

AntigenAB Ancell Cat Rev 2
Well Map MultiTope
Multi:Single 3 Plates

Single-Tope Plate Maps

The map of each Single-Tope plate is shown on the left.  The same capture plates are used for Multi-Tope and Single-Tope assays.  Captured EVs should be comparable between the two assays if the same EV preparation is used for both assays.

Each capture antibody has a performance data sheet available as described above.  By going to the Ancell Catalogue and searching the provided catalogue number, a pdf of the antibody performance data is available.

The Single-Tope assay kit comes complete with capture plates and all reagents needed to complete the assay except for a biotinylated detection antibody. This detection antibody is selected by the user.   An overnight capture followed by a 1 hour detection incubation yields a high definition fingerprint of the antigen profile of your EV preparation.

Use the Single-Tope assay to compare and contrast EV preparations from different sources, conditions, or production runs.  Identify unique antigens associated with your EV samples.  Follow multiple antigens on captured populations.  Screen with other antigen specific antibodies beyond tetraspans.

Single Tope 620 vs 480

Comparison of CD9 signal on specific antigen captured EVs.

EVix Single-Tope assay brings revolutionary screening power to EV biology.   Specific antigen capture yields different CD9 detection signals for different cell line supernatants.

Comparison of SW480 and SW620 cell derived EV preparations reveals significant differences in EV antigen profiles.  While both preparations have similar Class I antigen signals, the Class II signals suggest a absence of signal in SW620 when compared to SW480.

In contrast CD10 capture suggests that CD10 positive EVs are more prevalent in the SW620 preparation compared to SW480.

Singletope
20200529_111422

CD326 (EPCAM) On Urine Derived EVs

Specific antigen capture yields different antigen detection signals.

CD326 detected EVs are present only in the CD9 capture wells.  Captured CD326 positive EVs are not evident in the CD4, CD147, CD29 capture wells.  The absence of a CD326 detection signal in the CD326 capture well strongly suggests limited epitope availability for the CD326 antigen.

This Urine sample (100X) contained EVs that were captured by the different capture antibodies indicated.  This is indicated by the detection of either CD9, CD63, or CD81 antigens in the capture wells.

Urine Grph 1

Urine EV Antigen Patterns

Specific antigen capture yields different antigen detection signals.

CD326 capture again yields a CD9 detection signal but not for the other detection antigens.  CD10 capture EVs are negative for CD326 as expected from the CD326 capture and CD10 detection signal.

CD10 captured EVs do not yield CD24 or CD133 detection signals compared to CD13 detection for the same captured population.

This strongly suggests that on EVs isolated from urine, there is a difference in CD10 positive EVs compared to CD 133 positive EVs.  The different populations can be separated based on antigen detection capture strategies.

Urine 2 Grph
ELISA Plates Yellow

Urine EV Antigen Patterns

In continuation of the data above, an alternative depiction of results is shown using line graphing.  This is useful when examing many combinations of capture and detection antigens.

The green line represents signals generated by CD326 capture.  Again CD326 capture only yields a signal with CD9 detection.  This suggests that CD326 EPCAM bearing EVs do not express the other antigens.  Note that the signal rises above background only with CD9 detection.

The Dark blue line represents CD10 captured EVs.  Note sharp drop off of CD24 and CD133 detection signals.  The light blue line for CD133 capture indicates minimal CD10 detection signal, but a CD24 signal higher than any of the other captured populations.

All capture antigens however give strong CD9 detection signals, although CD326 capture is the least.  This indicates that CD9 tetraspan positive EVs were captured by the different capture wells.  Distinct differences in antigen distribution among the populations is readily apparent.

 

 

 

  • Screen more than 115 antigens in one assay

    Finger print EV populations for the presence and level of more than 115 unique and separate antigens in a 3 plate assay.

  • Reproducible

    Assay multiple samples with high sensitivity and reproducible results.  Compare Multi-Tope and Single-Tope results using the same capture plates.

  • High Resolution Finger Printing

    Use to compare EV preparations between populations and to qualify batch preparations.  Deep phenotyping of EV preparations can aid in assuring lot to lot similarities.

  • Efficient

    Run both negative controls and experimental values for every antigen.

  • Compare differences between EV populations

    Quantify unique antigen profiles in different samples and for different physiological conditions.  The absence or presence of antigens can be distinctive and related to EV sources and conditions.

  • Antigen Targets

    Select an antigen of interest and determine its distribution in your EV preparation. 

  • Build upon Multi-Tope Data

    The Multi Tope assay reveals antigens associated with the tetraspans CD9, CD63 and CD81.  Capture of EVs by the same Multi-Tope plates are now probed for expression of other non tetraspan antigens.

Note: Kit does not include a detection antibody.  It is recommended that a selected biotinylated Ab be sourced from Ancell Corp.

Kit Contents:  If contents are unopened and stored at 4o C the performance of the kit is stable for 6 months.

A: Single Tope Capture Plates:  Three (MT1, MT2 and MT3) 96 well plates containing 256 individual assay wells.  These are the same plates used in the Multi-Tope assay and Single-Tope data can be compared to results obtained in the Multi-Tope assay.   

B: 10X Sample and Wash Buffer Concentrate: 1 bottle (40 ml) to make 400 ml.  

C: Horse Radish Peroxidase Conjugated Streptavidin(SAHRP): 1 vial of 1.5 ml to make 30 ml.

D: Chromogenic Solutions : 1 bottle (30 ml) of a stabilized H2O2 , 1 vial (1.5 ml) of stabilized TMB chromogen Caution!

E: Stop Solution: 1 bottle (30 ml) Acid Caution! 

 

Order your Single-Tope assay kit from us (ST 001) and your detection antibody from Ancell.  Order them both today and they will arrive close to one another.  

Go to Ancell.com and order the detection antibodies of interest.  Ancell biotinylated antibodies are available in 100 ug vials and are recommended to be used at 2ug/ml concentrations.   One vial of biotinylated antibody is sufficient for 2 single tope assays.

SingleTope wout detect
EVix Colorimetric graphic

Catalog ST-001

256 Wells

$1395