EVix Selecta-Tope ELISA Kit
Research Use Only Not for Diagnostic Use
EVix Selecta-Tope ELISA kits are bespoke items designed by the researcher to address antigen specific capture and detect strategies in their EV research.
The Selecta-Tope ELISA kits are available in modular form. The capture module includes all components required to conduct a 96 well capture/detect assay including the reagents to create a coated capture plate. The capture module does not include capture antibody or detection antibody. These components are sold separately.
The amount of capture and detection antibodies available for purchase is as follows: A single capture antibody vial is sufficient to coat 2 capture modules ie. 2 (96 well) plates. The detection antibody is provided in an amount sufficient to assay 4 x 96 well plates. This allows flexibility for the researcher to mix and match both capture and detection antibodies and to acquire sufficient capture modules for the combinations selected. A single detection antibody vial, two capture antibody vials and 4 capture modules provides 4 complete 96 well assays with two unique capture antibodies.
Selected capture antibody and a module are used to create a functional capture plate. Provided with the capture module is the protocol and reagents needed to generate the specific capture plate. The process takes minutes to execute with several hours for incubation times, resulting in a capture plate with antigen specificity of choice. The capture module provides all necessary components to run the assay except capture and detection antibodies. It contains a streptavidin horse radish peroxidase reagent, a TMB chromogenic system, stop solution and a 10X wash buffer. The capture module with a selected capture antibody and selected detection antibody is custom tailored by the researcher for their particular needs.
The Selecta-Tope capture module incorporates individual 8 well strips providing the researcher the ultimate in flexibility in assay use and experimental design. Capture plates are readily created with all components needed provided in the kit. Once made, capture plates can be stored in the foil pouches at 4 C for many weeks prior to use. A single strip can be used independently leaving the remaining strips available for later use.
The Selecta-Tope kit builds upon data obtained via Multi and Single-Tope assays. It provides increased numbers of wells for evaluation of chromatography runs, or multiple replicates for more involved studies. It allows the researcher to focus on particular antigens deemed important in their studies, and lets them build a limitless combination of capture and detect capabilities. Additionally, if a particular detection antibody is not available from InCepix, they can be sourced from an alternative vendor and used with a capture antibody and capture module from InCepix. Lectins, or other detection systems can be used to probe captured EVs.
Capture plates can be used to deplete or reduce antigen specific populations from samples and thus used without detection antibody. These applications utilize capture modules and capture antibody only. These capture plates are used to capture and probe EV contents.
Selecta-Tope kits are flexible and specifc. Easy to order and to use. Try them today and realize the power of antigen capture and detection. Let us know your combination and we will help you in your order.
EVix Selecta-Tope ELISA Kit, when antigen specificity and high affinity are paramount in your EV experiments.
Track EV separations
Selecta-Tope assay allows tracking of chromatography EV separations. Comparison between different antigen capture strategies reveals presence of antigen in different sized EV preparations. Using Selecta-Tope assays, the elution profile of EV preparations can be tracked based on individual antigen profiles. Track tetraspans or other antigens by assaying column fractions. Chromatography provides additional insights into EV populations revealing presence of certain antigens in large and small EVs.
Separation of EVs by membrane filtration can also be used to select for distinct EV populations. Diafiltration against a 300,000 MWCO membrane concentrates both large and small EVs. Smaller EVs can be shown to cross a 500,000 MWCO membrane. This is confirmed by following antigen positive EVs as they elute off of a size exclusion columne. Larger EVs elute in the void volume, while smaller EVs show retention.
Track EVs by size and antigen.
Normal human Urine separated by diafiltration against a 500,000 MWCO membrane reveals EVs that pass through the membrane. When ran on a Sephacryl S500 HR (40,000 to 20,000,000 MW) chromatography column, these EVs are readily detected and reveal an elution pattern indicative of a fairly homogenous population of EVs. The 500,000 MWCO filtrate concentrated 200X against a 300,000 MWCO membrane reveals CD9 tetraspan positive species which can be captured by both CD26 and CD133 specific antibodies. The elution profile suggests that CD133 captured EVs may comprise 2 populations, in contrast to CD26 captured EVs. CD26 captured EVs clearly have CD10 antigen expression within their population.
EV Complexity Revealed by Antigen Tracking.
Culture supernatant collected and concentrated 100X by diafiltration against a 300,000 MWCO membrane reveals the heterogeneity in EV sizes. In contrast to the EVs which can pass through a 500,000 MWCO membrane, the concentrate against a 300,000 MWCO membrane reveals a much more heterogeneous population of EVs. Samples were run on Sephacryl S500 HR (40,000 to 20,000,000 MW). The blue line shows the elution profile of CD9 captured EVs detected by CD81. Note the heterogeneous elution profile of the tetraspan containing EVs. In contrast EVs captured by specific antigen (CD4, ClassI, ClassII, TCR VB1) and detected by CD9 positivity, demonstrate different elution profiles. Class I captured CD9 positive EVs over the entire elution volume. Smaller EVs captured by Class I and CD9 apear to have an overlapping elution profile while Class II, TCR VB1 and CD4 appear to be associated with primarily larger EVs. Shown below is supernatant from SW620 cell culture under the same conditions. In contrast, SW620 has a significant CD4 positive EV population which is heterogeneous in size, compared to SW480 cell culture supernatant. The Class I captured population is also heterogeneous in the SW620 population. CD10 positivity seen in the SW620 cell culture supernatants reflects the presence of Neprilysin positive EVs in SW620 but not SW480.
EV Antigen Capture and Depletion.
Selecta-Tope capture plates can deplete antigen specific EVs. SW620 cell culture supernatant (300,000 MWCO) was repetitively incubated on CD9 capture plates and detected with CD81 biotinylated detection antibody.
The axis marked capture round shows the results of repeated captures. A single capture round shows the signal after 2 hr capture incubation of signal. The well contents were transferred to a new capture well for an additional 2 hr incubation and its signal is shown as capture round 2. The fold dilution of EV antigen is the dilution of the starting antigen. The darker blue bars are the results for 100 fold dilution of the starting preparation. The orange bars are results for the 200 fold dilution.
After a single capture, the activity in the removed supernatant is reduced by more than 50% (compare blue bar to orange bar). The depletion is near complete after 2 rounds of capture for starting samples diluted 400X.
EV Antigen Capture Specificity.
SW620 culture supernatant derived EVs are captured by the indicated antibody and detected by CD9 detection antibody. In the checkered bars, the EVs were incubated with unlabeled antibody which competed with capture. CD24, CD4, CD10 and Class I captured EVs were specifically captured as shown and this capture was blocked by pre incubation with soluble antibody in 20 fold excess. EV sample was incubated separately in presence or absence of blocking antibody for 20 minutes prior to adding to capture well.
The Selecta-Tope kit provides the ability to analyze antigen specificity of capture by blocking/competition with matching antibody pairs. Each of the 115 antigens has both capture antibody as well as biotinylated and unlabeled antibodies available.
Profile EV Preparations with Combinations of Capture and Detection Antigens.
Normal human urine derived EVs (300,00 MWCO diafiltered) were captured by the indicated antibodies listed on top of chart. The captured EVs were than detected with the different detection antibodies shown on the bottom of chart. By plotting assay values linearly one can readily identify unique antigen distribution patterns.
Values at background and considered negative are readily seen when the line is approaching the bottom axis. For example ClassII detection of EVs was absent regardless of capture antigen. In contrast ClassI antigen was detected in several of the different antigen capture wells with CD147 antigen positve EVs yielding the largest Class I signal. and detected by CD9 detection antibody.
Select from 115 different capture antibodies and then pick out a biotinylated detection antibody to complete your kit. Bring your own capture antibody if so inclined and stock up on other capture antibodies.
Assay multiple samples with high sensitivity and reproducible results. Fully investigate antigen patterns revealed by the Multi-Tope and Single-Tope assays. 1 full capture/assay plate with modular 8 well strips containing antibody of choice along with all reagents to run complete assay.
High Resolution Finger Printing
Use to compare EV preparations between populations and to qualify batch preparations.
Capture wells are provided in modular 8 well strips. Coat all wells but use only the wells needed in your individual experiments. Stored capture wells are stable for months when stored in resealable foil pouch at 4 C.
Conduct in Depth Experiments
Run multiple replicates, dilutions, or processed samples. After phenotype analysis the Selecta-Tope Kits provide the tools needed to expand upon antigen specific experimental strategies.
Deplete or Remove EV populations
Capture wells can bind and remove EV populations from a sample. Multiple rounds of capture can significantly deplete antigen specific populations quickly and easily.
Kit Contents: A: EVix Capture Plate: (12 x 8 well strips) and frame. B: 10X Sample and Wash Buffer Concentrate: 2 bottles (10 ml each) to make 200 ml. C: Biotinylated Primary Detection Antibody: 1 vial of 0.5 ml to make 10 ml. D: Streptavidin Horse Radish Peroxidase SAHRP Conjugated: 1 vial of 0.5 ml to make 10 ml. E: Chromogenic Solutions : 1 bottle (10 ml) of stabilized H2O2 , 1 vial (0.5 ml) of stabilized TMB chromogen. Caution! F: Stop Solution: 1 bottle (10 ml) Acid Caution! G: EVTetraspan Standard: 1 vial 0.5 ml. If contents are unopened and stored at 4o C the kit is stable for 6 months.
Catalog SE 001