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, cancerology, infectious diseases, autoimmune diseases and transplantation.
The 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.
Principle of the method
After cell stimulation, locally produced cytokines are captured by IFN gamma and IL4 specific monoclonal antibodies. After cell lysis, trapped cytokine molecules are revealed by a secondary anti-IFN gamma FITC-conjugated antibody and a biotinylated anti-IL4 antibody. Those are in turn recognised by anti-FITC green fluorescent dye and streptavidin-phycoerythrin conjugates. PVDF-bottomed-well plates are then read under a UV light beam. Green fluorescent spots indicate IFN gamma production while IL4 is revealed by red spots. Yellow spots will indicate dual cytokine producing cells.
Plates should be stored at room temperature.
|Components||10 x 96 tests|
|96 PVDF-bottomed-well plates||1 unit|
|Anti-FITC antibody green fluorescence conjugate.||1 vial|
|Biotinylated detection antibody for IL-4 ( resuspend in 0.55mL).||2 x 0.55ml|
|Bovine Serum albumin.||2 x 1g|
|Capture antibody for IFNg||2 x 0.5ml|
|Capture antibody for IL-4||2 x 0.5ml|
|FITC conjugated detection antibody for IFNg (resuspend in 0.55mL).||2 x 0.55ml|
|Fluorescence buffer||2 x 2.5ml|
|Streptavidin-phycoerythrin conjugate.||1 vial|
Our Abpromise guarantee covers the use of ab46647 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|FluroSpot||Use at an assay dependent dilution.|
ab46647 has not yet been referenced specifically in any publications.