Sandwich Elisa Assay

Sandwich Elisa Assay Method

A description of the sandwich Elisa assay method including a diagram and protocol.

Summary of the Sandwich Elisa assay:

Step 1 ) The microwell plate (usually 96-well) is coated with a capture antibody. This has been previously been shown to bind to your antigen of interest, either through western blot assays or other detection methods.

Step 2) Your biological sample is added to the microplate well. Any antigen present is bound to the capture antibody which is coated on the well. Non-specific proteins or unbound proteins are usually washed off.

Step 3) The detecting antibody is added to the well and binds to the antigen, creating a "sandwich" hence the name "sandwich" ELISA.

Step 4) Enzyme-linked antibody is added to the wells, and binds to detecting antibody. Enzyme-linkede meaning a protein capable of changing a substrate to a form detectable by a plate reader ie color or light change. This is where ELISA gets the EL (enzyme-linked)

Step 5) Chromogenic or light-forming substrate is then added. The substrate is converted by enzyme to the detectable form.

Step 6) Amount of color or light-change is detected by a plate reader and quantified. The amount of light / color is proportional to the amount of antigen you had in your biological sample which you added to that well.