EVix Selecta-Tope ELISA Kit
Research Use Only Not for Diagnostic Use
The Selecta-Tope capture module includes all components except capture and detection antibody, required to conduct a 96 well capture/detect assay. This allows for custom capture and detection strategies and flexibility in experimental design.
Capture and detection antibodies available for purchase from Ancell are easily combined with the capture module assay kit. A single 100 ug vial of capture antibody is sufficient to coat 2 capture modules. The 100 ug vials of detection antibody are sufficient to assay 4 modules. This allows the researcher to mix and match both capture and detection antibodies. A single detection antibody vial, two capture antibody vials and 4 capture modules provides 4 complete 96 well assays with two unique capture antibodies.
Provided with the capture module are reagents needed to generate the antigen 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 also provides all necessary components to run the assay. 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 and detection antibody is a custom tailored solution dedicated to researcher needs.
The Selecta-Tope kit builds upon data obtained via Multi-Tope and Single-Tope assays. It provides increased numbers of wells for evaluation of chromatography runs, or multiple replicates. 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. 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.
Capture plates can also be used to deplete or reduce antigen specific populations from samples and used without detection antibody. These applications utilize capture modules and capture antibody only.
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.
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. EVs captured with anti Class I wells, were CD9 positive 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 Demonstrated by Antibody Blocking
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 EV 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 anti MHC Class II detection was absent regardless of capture antigen. In contrast Class I antigen was detected in several of the different antigen capture wells with CD147 antigen captured EVs having the largest Class I signal.
Customize capture and detection strategies to track unique EV populations.
Capture unique EV populations using selected capture and detection antibodies.
Design capture plates selecting from specific antigens of relevance.
Track EVs from specific sources by focusing on unique antigen profiles. Discriminate between EV populations by tracking specific antigens.
Create and store capture plates for multiple detection antigens
Prepare capture plates and store until use. Use your own antigen specific antibody or obtain Ancell antibodies ready for coating.
Run only wells needed from modular plates. Build up capture modules with all needed components to run a complete assay. Just add your selected antibodies, all other components provided.
Compare differences between EV populations
Quantify unique antigen profiles associated between different samples and clinical conditions. The absence or presence of antigens can be distinctive and related to EV sources.
Select the antigen of interest for both capture and detection. Capture antigen positive EVs and probe for selected antigen of choice.
Build upon Multi-Tope Data
The Multi Tope assay will reveal antigens associated with the tetraspans CD9, CD63 and CD81. Capture of EVs by the Multi-Tope antigens are then probed for expression of other non tetraspan antigens. Antigen combinations can than be focused on by building capture and detect specific plates.
Kit Contents: A: EVix Capture Plate: (12 x 8 well strips) and frame. B: 10 ml Coating Buffer. C: 10X Sample and Wash Buffer Concentrate: 2 bottles (10 ml each) to make 200 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!
If contents are unopened and stored at 4o C the kit is stable for 6 months.
- Order SE-001 for Capture Module
- Does not include Capture Antibody
- Does not include Biotinylated Detection Antibody