ELISA (Enzyme-linked Immunosorbent Assay) detects particles with a simple color change by using antibodies. When antibodies are exposed to their corresponding antigen, it will bind to it to instigate an immune response. Researchers can utilize this ‘hook-like’ mechanism to detect particles like peptides, proteins, and hormones. There are also a variety of ELISA types, such as Direct or Indirect, Sandwich, and Competitive ELISA. Each of these have their own applications, allowing ELISA to be used for various tests, especially for medical reasons or toxicology reports.
Simply put, what is required for an ELISA test, in general, are wells with which contain an antigen, detection enzymes, antibodies, and your sample. Although some types of ELISA work in different ways, the underlying principle always remain the same: the immobilization of an antigen. Modifications on this theme, like whether the antigen is caught with an antibody attached to a plate or suspended in solution, is what makes ELISA so useful for detection. Once particles of interest are stuck onto the ELISA plate, then all that is necessary is to wash the plate’s surface, separating attached from unattached particles.
To make researchers lives easier, detection enzymes (or any other tag) can be attached to primary or secondary antibodies, or proteins. Once a detection enzyme encounters its corresponding substrate, light is usually emitted and measured depending on your experimental setup (fluorometer, luminometer, spectrophotometer).
Our ELISA Troubleshoot provides further assistance.
Types of ELISA
Direct ELISA - Refers to direct detection; a primary antibody (labeled with a detection enzyme), reacts directly with an antigen, which is immobilized on the plate.
Indirect ELISA – Indirect detection; a labeled secondary antibody with known specificity for a primary antibody
Sandwich ELISA – one of the most common methods of ELISA, and the steps are as follows:
Capture antibody is placed onto a plate
Sample antigen is added, binding to the capture antibody
Detection antibody is introduced, binding also to the antigen
Secondary antibody with a detection enzyme is added, binding to the detection antibody
Substrate is added, thus triggering all successfully captured antigens within sample
Sample antigen is incubated with complementary unlabeled
The antibody/antigen complexes are inserted into a well coated with competitive antigen.
Washing the plate removes unbound antibodies. (The higher the volume of antigen in the sample, the more Ag-Ab complexes present and therefore less unbound antibodies free to bind to the antigen coating the well)
Secondary antibody coupled to an enzyme is added, which binds the primary antibody.
Adding the substrate for the enzyme produces a chromogenic or fluorescent signal, depending on the reaction
Stopping the reaction prevents the signal from saturating
Enzyme-linked antigen can be used in place of enzyme-linked antibody. In this case labeled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). A stronger signal is seen if the there is less antigen in the sample, as more labeled antigen is retained in the well.
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