The ELISA Method
|The ELISA technique illustrated|
Coat plate with capture mAb
Add samples and standard
Add biotinylated detection mAb
Add chromogenic substrate for color development
ELISA Assay Procedure
The cytokine ELISA (Enzyme-Linked Immuno Sorbent Assay) is a specific and highly sensitive method for quantitative measurements of cytokines or other analytes in solutions.
A specific monoclonal antibody (mAb) able to capture the cytokine of interest is coated on a microtiterplate. A second mAb, used for detection, binds a different epitope on the cytokine. The detection mAb is labeled with biotin, which allows subsequent binding of a Streptavidin-conjugated enzyme. Any unbound reagents are washed away.
When substrate is added, a color reaction will develop that is proportional to the amount of cytokine bound. The concentration of cytokine is determined by comparison with a standard curve with known concentrations of cytokine.
The sensitivity of an ELISA depends mainly on the affinity of the antibodies and on the amplification system used.
The detection limits for cytokine ELISAs are commonly in the lower picogram/ml range.