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.
Principle of Method
After cell stimulation, locally produced cytokines are captured by IL-10 and IL-2 specific monoclonal antibodies. After cell lysis, trapped cytokine molecules are revealed by a secondary anti- IL-10 FITC conjugated antibody and a biotinylated anti-IL-2 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 IL-10 production while IL-2 is revealed by red spots. Yellow spots will indicate dual cytokine producing cells.
|Components||5 x 96 tests||10 x 96 tests|
|96 PVDF-bottomed-well plates.||5 units||10 units|
|Anti-FITC antibody green fluorescence conjugate.||1 vial||2 vials|
|Biotinylated detection antibody for IL-2 (resuspend in 0.55mL).||1 vial||2 vials|
|Bovine Serum albumin.||1 x 1g||2 x 1g|
|Capture antibody for IL-10||1 x 500µl||2 x 500µl|
|Capture antibody for IL-2||1 x 500µl||2 x 500µl|
|FITC conjugated detection antibody for IL-10(resuspend in 0.55mL).||1 vial||2 vials|
|Fluorescence buffer||1 x 2.5ml||2 x 2.5ml|
|Streptavidin-phycoerythrin conjugate.||1 vial||2 vials|
Our Abpromise guarantee covers the use of ab48460 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.|
ab48460 has not yet been referenced specifically in any publications.