In the ELISA test, what do primary antibodies & secondary antibodies do?
1 Answer
That depends on the type of ELISA assay.
Explanation:
ELISA = Enzyme Linked Immunosorbent Assay.
There are different types of ELISA assays. What you at need is:
- antibody with an enzyme attached to it
- antigen to which the antibody binds
- substrate of the enzyme which causes a measurable reaction
The enzyme attached to the antibody is often HRP (horseradish peroxidase). HRP can react with different substrates that either changes color or emits light, both measurable.
The image shows an overview of the different types of ELISA assays. I will explain how they work below.
Direct ELISA
A plate is coated with the antigen (Ag) and a primary antibody with enzyme is added. When the substrate is added, the reaction will occur and is proportional to the amount antibody-antigen interactions.
Indirect ELISA
Again, a plate is coated with the antigen and a primary antibody binds to the antigen. A secundary antibody is added that binds to the first antibody. The 2nd antibody contains the enzyme that reacts with the substrate.
Sandwich ELISA
Here two or three antibodies are involved. A capture antibody is attached to plates and binds to the antigen. This is followed by the same steps as either the direct (only one antibody) or indirect method (two antibodies).
Competitive ELISA
Sometimes called inhibition ELISA, is a bit more complicated. A plate is coated with the antigen to be researched. A primary antibody with enzyme is first incubated with a sample with an unknown concentration of the same antigen. This is then added to the plate, allowing the antibodies that are still free to react with the antigen on the plate. After the antibody-antigen complexes of the sample are washed off the substrate is added.
When the unknown sample has a lot of antigen, there will be less antibody free to bind to the plate = lower signal.
