Enzyme-Linked Immunosorbant Assay (ELISA)
ELISAs allow for sensitive, highly specific, and even quantitative detection of antigen in different types of samples. A host of variations exist on the procedures commonly used to perform these assays and the images below illustrate two common variations and the fundamental concepts behind ELISA assays.
Indirect ELISA - A solution containing purified or unpurified antigen (Ag) is incubated in a microtiter well under conditions where some of the antigen spontaneously adheres to the solid surfaces (e.g. by electrostatic adherence to the plastic wells of a microtiter plate). For quantitative comparisons, a range of Ag concentrations is usually introduced into other wells in the same experiment. After incubating and washing away unbound Ag, monoclonal or polyclonal first antibody (1st Ab) is added. After washing away unbound 1st Ab, a second antibody with an enzyme (E) covalently conjugated to it (2nd E-Ab) is added. This antibody specifically reacts against different portions of the same Ag. One of many possible enzymes that might be covalently attached is horse radish peroxidase (HRP). After incubating and washing away unbound 2nd E-Ab, a chemically reactive substrate (S) is added such that the remaining enzyme in the well will catalytically convert it into an easily detectable colored, fluorescent, or luminescent product. The resulting intensity of color, fluorescence, or luminescence will be indirectly proportional to the amount of Ag remaining in the reaction well.
IMMEX/Sandwich ELISA - A solution containing a monoclonal or polyclonal first antibody (1st Ab) is incubated in a microtiter well under conditions where some of the antibody spontaneously adheres to the solid surfaces (e.g. by electrostatic adherence to the plastic wells of a microtiter plate). After incubating and washing away unbound 1st Ab, a solution containing purified or unpurified antigen (Ag) is added. For quantitative comparisons, a range of Ag concentrations will be introduced into other wells in the same experiment. After washing away unbound Ag, a second antibody with an enzyme (E) covalently conjugated to it (2nd E-Ab) is added. This antibody specifically reacts against different portions of the same Ag. One of many possible enzymes that might be covalently attached is horse radish peroxidase (HRP). After incubating and washing away unbound 2nd E-Ab, a chemically reactive substrate (S) is added such that the remaining enzyme in the well will catalytically convert it into an easily detectable colored, fluorescent, or luminescent product. The resulting intensity of color, fluorescence, or luminescence will be indirectly proportional to the amount of Ag remaining in the reaction well.
