Competitive Elisa Assay

Competitive Elisa Assay Method

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

Summary of the Competitive Elisa assay:

The steps for competitive ELISA assay include

Step 1. Unlabeled primary detecting antibody is added to a biological sample and incubated in the presence of its antigen.

Step 2. The bound antibody-antigen complexes and unbound antibodies are then added to an microplate well which is coated with the antigen of interest.

Step 3. The plate is washed such that unbound antibody is removed from the well. Therefore, the more antigen you had in your biological sample, the less antibody that will be present to be able to bind to the antigen in the well and create a signal. Hence the term "competition" in competitive ELISA.

Step 4. Secondary antibody which is specific to the primary 1o antibody is added to the well. This secondary antibody is linked to an enzyme which can cleave a substrate into a detectable form i.e either creates color or light.

Step 5. Substrate is added to the well and the bound antibody-enzymes create either chromogenic or fluorescent signal which is detectable by a plate reader.

 

In summary, Competitive ELISA : the weaker the signal, the higher the original antigen concentration was.