EVix Multi-Tope ELISA Kit 256 Wells/115 Ab
Research Use Only Not for Diagnostic Use
Description: EVix Multi-Tope ELISA kit brings revolutionary screening capabilities to EV assays. The Multi-Tope assay, uses highly specific monoclonal antibodies to capture antigen specific EVs. Captured EVs are detected with an antibody mixture specific to CD9, CD63, and CD81. Expression of multiple tetraspan molecules on single EVs, allows even small numbers of captured EVs to be detected and quantified. The high sensitivity of the Multi-Tope assay can detect antigens that are present at low levels in an EV population.
The Multi-Tope ELISA kit contains capture antibody in duplicate wells, one to capture EVs from your sample and the other as a negative control to track background detection antibody binding. The signals generated are thus specific for EVs captured with the unique capture antibody within the individual well.
The Multi-Tope assay has unique capture plates containing 115 unique capture antibodies individual wells. The complete assay kit allows assessment of 115 unique antigens in an EV sample along with background signal in the absence of EVs.
All capture antibodies are certified and coated at optimum concentrations to yield readable signals. The capture monoclonal antibodies are selected from our catalogue of surface and immune associated antigens. Each antibody has been tested and qualified by flow cytometry and additional information is provided by the manufacturer for each clone. Please reference the Ancell catalogue online for information on each clone, including a performance data sheet of each antibody's performance. The Ancell data can be searched by catalogue number given for each antigen specific clone below.
The Multi-Tope ELISA kit comes complete with all reagents needed to run the 256 well assay. In contrast to a 2 hour capture for the HSTT assay, 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 CD9, CD63 and or CD81.
Multi-Tope Plate Maps
The map of capture antibodies is shown on left. There are 2 complete plates and 1 partial plate. A total of 115 unique capture antibodies with additional control anti tetraspan (CD9, CD63 and CD81) antibodies are provided.
Paired wells provide the negative control well next to the experimental. Odd columns do not receive antigen only sample/wash buffer and provide a non specific background signal which is subtracted from the experimental well containing the EV preparation.
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 for download.
The Multi-Tope assay kit comes complete with capture plates and all reagents needed to complete the assay. An overnight capture followed by a 1 hour detection incubation yields a high definition fingerprint of the antigen profile of your EV preparation.
Use to compare and contrast EV preparations from different sources, conditions, or production runs. Identify unique antigens associated with your EV samples.
Finger Print your EV preparations.
The EVix Multi-Tope assay brings revolutionary screening power to EV biology. Each assay screens over 100 unique cell surface antigens. If the target antigen is present, the EV is captured. The detection antibody cocktail of anti CD9, CD63, and CD81 antibodies mazimizes signal and specific antigen bearing EVs are detected. There is no multiplexing or flow cytometry involved. Add sample to the Multi-Tope wells and capture antigen specific EVs overnight.
Unlike Mass Spectrometry, the Multi-Tope assay reads only membrane/tetraspan associated signals captured by specific antigen. Hundreds of antigens are reportedly EV associated, however many of these studies run entire preparations of EVs that have been concentrated by centrifugation or precipitation and co-seperated molecules can lead to detection of molecules not necessarily associated with membranous vessicles.
The Multi-Tope assay screens your EV preparations quickly and thoroughly creating a unique phenotype. Much like a finger print, the Multi-Tope assay can be used to confirm similarities or differences between EV preparations.
Screen for Antigen Presence.
The EVix Multi-Tope assay brings revolutionary screening power to EV biology. 13 ml of EV preparation adjusted to give a HSTT value of 3 or higher yields antigen profiles specific to the preparation.
Negative control signals are also shown, which are background signals without EV sample. This sample is normal human urine, concentrated 100X by diafiltration against a 300,000 MWCO membrane. Samples were dia filtered against TBS glycine solution. Note presence of EPCAM (CD326) on tetraspan specific EVs obtained from normal unringe. Neprilysin (CD10) is also present on urine EVs.
Phenotype EV Preparations.
Antigen signals vary significantly between EV origin. The panel on the left shows results from a human mixed lymphocyte cultrue supernatant harvested on Day 7 of culture.
In contrast to the urine antigen patterns, the EV phenotype obtained from a mixed lymphocyte culture sample shows the presence of more immune associated molecules.
Negative control signals are not shown but have been subtracted out from the positive values. This sample is culture supernatant that has been concentrated by diafiltration in TBS glycine against a 300,000 MWCO membrane.
Rank Antigen Signals.
Antigen signals not only vary between antigen preparations from different sources, but also within preparations. Since the antigen signal is quantitative, the antigen specific signals can be ranked and plotted according to intensity. As shown left, the signals from pooled culture supernatants run at 50X concentration have a hierarchy and can be used to further characterize EV populations.
Negative control signals are not shown having been subtracted out from the positive values. This sample is a pool of different cell culture supernatants that has been concentrated by diafiltration in TBS glycine against a 300,000 MWCO membrane.
Compare and Contrast EV Populations.
Shown left is an XY scatter plot of antigen signals for a Urine sample (Horizontal) and mixed culture supernatant (Vertical) obtained by Multi-Tope assay.
While the antigens in the upper right corner appear to be shared in both preparations, there are distinct antigens present only in one of the preparations, and fall close to the axis. The antigens shared are tetraspan like and integrin related. Antigens unique to the preparation are other cell surface antigens as indicated.
Screen More Than 115 antigens In One Assay
Finger print tetraspan positive EV populations for presence and level of more than 115 unique and separate cell surface antigens in a 3 plate assay.
Assay multiple samples with high sensitivity and reproducible results.
High Resolution Finger Printing
Use to compare EV preparations between populations and to qualify batch preparations. Captured antigen specific EVs are detected by a triple detection antibody cocktail. High sensitivity is achieved by detecting CD9, CD63 and CD81 simultaneously on captured EVs.
Run both negative controls and experimental values for 115 antigens in a single assay. Identify unique antigen profile for your EV preparations.
Compare Differences Between EV Populations
Characterize antigen profiles of different samples and clinical conditions. The absence or presence of antigens can be distinctive and related to EV sources.
Antigen Specific Capture
Wells of Multi-Tope assay plates are coated with unique monoclonal capture antibodies. The presence of a signal from the triple antibody detection cocktail indicates that the capture antigen is present along with either CD9, CD63, or CD81 tetraspans.
Multi-Tope Phenotype Data
The Multi Tope assay reveals antigens associated with the tetraspans CD9, CD63 and CD81. Different EV populations can have different Multi-Tope antigen profiles. This can be used to phenotype your EV preparations and comparing them to other populations.
A: Multi Tope Capture Plates: Three (MT1, MT2 and MT3) 96 well plates (Store 4C) containing 256 individual assay wells
B: 10X Sample and Wash Buffer Concentrate: 1 bottle (40 ml) to make 400 ml. (Store 4C)
C: Biotinylated Primary Detection Antibody Cocktail: 1 vial of 1.5 ml to make 30 ml. (Store -70C)
D: Horse Radish Peroxidase Conjugated Streptavidin(SAHRP): 1 vial of 1.5 ml to make 30 ml. (Store -70C)
E: Chromogenic Solutions : 1 bottle (30 ml) of a stabilized H2O2 , 1 vial (1.5 ml) of stabilized TMB chromogen (Store 4C) Caution!
F: Stop Solution: 1 bottle (30 ml) Acid Caution! (Store RT)
If contents are unopened and stored at 4C and -70C the performance of the kit is stable for 6 months.
- Order MT-001.
- Complete kit for 256 well assay.
- 115 unique capture antibodies