High Sensitive IFN gamma ELISA Assay

Human sCD25 ELISA Assay

$635.00

The Eagle Biosciences sCD25 ELISA kit is a solid phase sandwich ELISA for the in-vitro qualitative and quantitative determination of sCD25 in supernatants, buffered solutions or serum and plasma samples. This assay will recognise both natural and recombinant human sCD25. The Eagle Biosciences Human sCD25 ELISA Assay kit is for Research Use Only.

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Human sCD25 ELISA Assay

The Human sCD25 ELISA Assay is For Research Use Only

Size: 1×96 wells
Sensitivity: 32.5 pg/ml
Dynamic Range: 68.75 pg/ml – 2220 pg/ml
Incubation Time: 3h 40min
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Names: IL-2R, Interleukin 2 Receptor, soluble IL-2R

Assay Background

IL-2, one of the most important factors in the human immune system, is a potent T-cell growth factor whose major function is the activation of many cells of the immune system including T-cells, B-cells, macrophages and NK cells. These potent actions are mediated by IL-2 binding and signalling through its associated cell surface receptor IL-2R. This receptor is not expressed on normal or unstimulated lymphocytes but is quickly transcribed and expressed on T-cells following activation.

This IL-2R is a heterotrimeric protein consisting of three distinct glycopeptide subunits termed IL-2Ra (CD25) specific to IL-2R, IL-2Rb and IL-2Rg. The a and b chains are involved in binding IL-2 while the signal transduction following IL-2 binding is mediated by the g-chain along with the b chain. The IL-2Ra chain or CD25 is a type 1 transmembrane glycoprotein of 251 amino acids and 55kDa. CD25 can also be found as a soluble form in serum and tissue following enzymatic cleavage from expressing cells and can be identified as a 45KDa protein once shed from the membrane. As the expression and subsequent release of CD25 takes place following cell stimulation the presence of soluble CD25 (sCD25) in circulation is an excellent marker of T-cell activation.

A number of disease states linked to over expression of CD25 have previously been described including, autoimmune diseases, transplant rejection, chronic infection, B-cell neoplasm and various types of leukaemia and other forms of cancer. Because of this definite link between CD25 over expression and disease state many therapies for these conditions have evolved to inhibit this over expression of IL-2Ra.

More recently CD25 has become the major marker for distinguishing the CD4+CD25+ subset of T regulatory cells.

Related Products

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Human CD124 ELISA Assay

Additional Information

Assay Principle

A capture Antibody highly specific for CD25 has been coated to the wells of the microtitre strip plate provided during manufacture. Binding of CD25 samples and known standards to the capture antibodies and subsequent binding of the biotinylated anti-CD25 secondary antibody to the analyte is completed during the same incubation period. Any excess unbound analyte and secondary antibody is removed. The HRP conjugate solution is then added to every well including the zero wells, following incubation excess conjugate is removed by careful washing. A chromogen substrate is added to the wells resulting in the progressive development of a blue coloured complex with the conjugate. The colour development is then stopped by the addition of acid turning the resultant final product yellow. The intensity of the produced coloured complex is directly proportional to the concentration of CD25 present in the samples and standards. The absorbance of the colour complex is then measured and the generated OD values for each standard are plotted against expected concentration forming a standard curve. This standard curve can then be used to accurately determine the concentration of CD25 in any sample tested.

  1. Add 100μl of each, Sample, Standard, Control and zero  in duplicate to appropriate number of wells
  2. Add 50μl of diluted biotinylated anti-CD25 to all wells
  3. Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 3 hour(s)
  4. Remove the cover and wash the plate.
  5. Add 100μl of Streptavidin-HRP solution into all wells
    Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) for 30 min
  6. Repeat wash step 4.
  7. Add 100μl of ready-to-use TMB Substrate Solution into all wells
  8. Incubate in the dark for 10-15 minutes* at room temperature. Avoid direct exposure to light by wrapping the plate in aluminium foil.
  9. Add 100μl of H2SO4: Stop Reagent into all wells

Typical Standard Curve

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