ELISA: A Step By Step Method Guide
ELISA Biological Method Overview
ELISA is the common acronym for Enzyme-Linked-Immunosorbent Assay. It’s a quick plate based technique for detecting an antigen from a solution. This antigen could be a peptide, protein, antibody, or small molecule. In general, for an ELISA, an antigen is first immobilized on a surface (Step 1 below). Next, an antibody specific to the antigen is flowed over the surface (Step 2). This antibody, is also attached to a chemiluminescence-related enzyme. Treatment with the chemiluminescent substrate facilitates detection of the antibody and the antigen (Step 3). Take a look at these pictures to get an overview of the strategy:
Conjugated proteins may require different antibodies in ELISAs. However you can easily track the ELISA process by labeling your protein with a fluorescent probe.
Types of ELISAs
There are a few different types of ELISA assays but they all follow the basic strategy outlined above. Essentially, one can choose how to immobilize the antigen on the surface and how the antigen is detected via the antibody.
- Direct Assay: In this method, the antigen is immobilized to the surface and detected directly via an antibody that’s bound to a chemiluminescent enzyme. (Same as above)
- Indirect Assay: In this method, the detecting antibody doesn’t have the chemiluminescent enzyme. So, another antibody must bind to the first antibody to facilitate detection.
- Sandwich Assay: The most common type of ELISA. In this assay, a “capture” antibody is first immobilized to the substrate. Then antigen is flowed over it so that it gets immobilized to the surface along with the capture antibody. Finally the detection antibody is flowed over the substrate and it binds the antigen. This detection antibody may be directly conjugated to the chemiluminescent enzyme (just like a direct assay) or another antibody may be needed (just like the indirect assay).