Human IL-2 HS ELISA Assay

Additional Information

Assay Principle

The IL-2 Kit is a solid phase sandwich Enzyme Linked-Immuno- Sorbent Assay (ELISA). A monoclonal antibody specific for IL-2 has been coated onto the wells of the microtiter strips provided. Samples, including standards of known IL-2 concentrations and unknowns are pipetted into these wells.  During the first incubation, the IL-2 antigen and a biotinylated monoclonal antibody specific for IL-2 are simultaneously incubated.

After washing, the enzyme (streptavidin-peroxydase) is added. All the unbound enzyme is removed by washing and the first amplification step is performed by adding the Biotine-Tyramine reagent. Under the action of HRP, a biotine polymerisation reaction occurs in the region of the HRP linked to the detection antibody. After washing the second amplification step is performed and the polymerised biotine is revealed by a new streptavidin-HRP step. Finally after washing, the substrate is added. The intensity of this coloured product is directly proportional to the concentration of IL-2 present in the samples.

Assay Procedure

1. Add 100μl of each standard, sample and zero (appropriate Standard Diluent Buffer) in duplicate to appropriate number of wells
2. Add 50μl of diluted biotinylated anti-IL-2 to all wells
3. Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) with slow shaking for 1 hour
4. Remove the cover and wash the plate
5. Add 100μl of Streptavidin-HRP solution 1 into all wells
6. Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) with slow shaking for 20 min
7. Repeat wash step 4.
8. Add 100μl of diluted Amplifier to all wells
9. Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) with slow shaking for 15 min
10. Repeat wash step 4.
11. Add 100μl of Streptavidin-HRP solution 2 into all wells
12. Cover with a plastic plate cover and incubate at room temperature (18 to 25°C) with slow shaking for 20 min
13. Repeat wash step 4.
14. Add 100μl of ready-to-use TMB Substrate Solution into all wells
15. Incubate in the dark for 10-20 minutes* at room temperature. Avoid direct exposure to light by wrapping the plate in aluminium foil
16. Add 100μl of H2SO4:Stop Reagent into all wells

Typical Standard Curve

Specificity

Ten specificities were tested with concentrations higher than IL-2 curve concentrations in the Human High Sensitivity IL-2 ELISA Assay Kit.  No cross reaction was observed for concentrations ranging from 300 to 18.75 pg/ml for IL-1α and β, IL-8, IL-10, TNF-α, GrB, FasL, TRAIL, CD116 and Gp-130.