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The ELISPOT assay is designed to enumerate cytokine producing cells in a single cell suspension. This method has the advantage of requiring a minimum of in-vitro manipulations allowing cytokine production analysis as close as possible to in-vivo conditions in a highly specific way. This technique is designed to determine the frequency of cytokine producing cells under a given stimulation, and the follow-up of such frequency during a treatment and/or a pathological state. Elispot assay constitutes an ideal tool in the TH1 / TH2 response, vaccine development, viral infection monitoring and treatment, oncology, infectious diseases, autoimmune diseases and transplantation.
This Elispot assay is based on sandwich immuno-enzyme technology. Cell secreted cytokines or soluble molecules are captured by coated antibodies avoiding diffusion in supernatant, protease degradation or binding on soluble membrane receptors. After cell removal, the captured cytokines are revealed by tracer antibodies and appropriate conjugates.
The dual colour Elispot allows you to monitor the production of two cytokines simultaneously in the same well.
Principle of the Method
After cell stimulation, locally produced cytokines are captured by IFN gamma and Granzyme B specific monoclonal antibodies. After cell lysis, trapped cytokine molecules are revealed by a secondary anti-IFN gamma FITC conjugated antibody and a biotinylated anti-Granzyme B antibody. Those are in turn recognised by anti-FITC HRP and streptavidin-AP conjugates. PVDF-bottomed-well plates are then incubated first with AEC substrate buffer, washed and subsequently incubated with BCIP/NBT. Coloured red/brownish spots indicate IFN gamma production while Granzyme B is revealed by blue/purple spots.
|Components||5 x 96 tests||10 x 96 tests|
|10 x concentrate buffer for the preparation of AEC buffer||1 x 5ml||2 x 5ml|
|50 x concentrate AEC substrate buffer||1 x 1ml||2 x 1ml|
|96 PVDF-bottomed-well plates.||5 units||10 units|
|Anti-FITC antibody HRP conjugate||1 x 100µl||2 x 100µl|
|Bovine Serum albumin||1 x 1g||2 x 1g|
|Dry skimmed milk||1 x 1g||2 x 1g|
|Granzyme B Biotinylated detection antibody||1 vial||2 vials|
|Granzyme B Capture Antibody||1 x 500µl||2 x 500µl|
|Human IFNγ Capture antibody||1 x 500µl||2 x 500µl|
|IFNγ FITC conjugated detection antibody||1 vial||2 vials|
|Ready-to-use BCIP/NBT substrate buffer||1 x 50ml||2 x 50ml|
|Streptavidin - Alkaline Phosphatase conjugated||1 x 50µl||2 x 50µl|
Our Abpromise guarantee covers the use of ab48464 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ELISpot||Use at an assay dependent dilution.|
ab48464 has not yet been referenced specifically in any publications.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"