Thinking about ELISAs? Well, you better make sure you can talk the talk before you start to walk the walk. You know, learn the ELISA lingo.
Here are the basic terms you should know:
The process of adsorbing the antigen or antibody to the plate is often called “coating.”
This is also called the adsorption step.
Passive adsorption occurs when the plate is coated simply by incubating the wells of the plate with antigen or antibody, which randomly bind to the plate in any orientation.
While passive adsoprtion is quick, it can decrease the sensitivity of the ELISA as some of the bound protein may not be able to correctly present an epitope for antibody binding.
Direct detection method
The detection method you are using when your primary antibody is directly labeled with the enzyme or dye.
Direct detection requires fewer steps, but can be less sensitive.
Indirect detection method
The detection method you are using when your primary antibody is unlabeled and you use a labeled secondary antibody to recognize the primary antibody.
Indirect detection is more flexible and more sensitive.
A modification of the standard ELISA assay that increases the specificity of the assay. In a sandwich ELISA the plate is first coated with an antibody specific for the antigen of interest, which binds the antigen on the plate when exposed to the sample. A second antibody that recognizes the same antigen is then used to detect the bound protein.
It’s an antibody sandwich with a layer of target protein in between.
The antibody that is used to coat the plate in a sandwich ELISA.
It “captures” the antigen.
The antibody that is used to detect the antigen in a sandwich ELISA. If you are doing a direct sandwich ELISA, then you will use one detecting antibody. If you are doing an indirect sandwich ELISA then you will use two detecting antibodies – the primary antibody and the labeled secondary antibody.
An automated machine that efficiently and accurately washes ELISA plates in between incubations.
If you are going to be doing a lot of ELISAs, you should make friends with a lab that has one of these!
Photo courtesy of Alan Levine.