ELISA: A Step By Step Method Guide
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 ELISA, the antigen is first immobilized on the surface.
The antigen-specific antibody is then poured onto the surface. These antibodies are also associated with enzymes associated with chemiluminescence. Treatment with a chemiluminescent substrate facilitates the detection of antibodies and antigens. Take a look at these photos to get an overview of the strategy:
Conjugated proteins may require different antibodies in the ELISA. However, you can easily monitor the ELISA process by labeling your protein with a fluorescent probe. There are many companies available that provide ELISA kits online. You can easily get the PLAT elisa kits online from various online sources.
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There are several types of ELISA assays, but all follow the basic strategy outlined above. Basically, one can choose how the antigen is immobilized on the surface and how the antigen is detected by the antibody.
1. Direct analysis: In this method, the antigen is immobilized on the surface and directly detected by the antibody bound to the chemiluminescent enzyme. (Same as above)
2. Indirect analysis: With this method, the detecting antibody does not contain chemiluminescent enzymes. Therefore, another antibody must bind to the first antibody to facilitate detection.
3. Sandwich analysis: The most common type of ELISA. In this assay, the "capture" antibody is first immobilized on the substrate. The antigen is then poured to immobilize the surface along with the captured antibody.
4. Finally, the detecting antibody is transferred to the substrate and binds to the antigen. These detecting antibodies may be conjugated directly to the chemiluminescent enzyme (such as direct testing) or other antibodies may be required (such as indirect testing).