ELISA is a biochemical or biological technique used to detect the presence of certain components in a sample. Depending on the type of ELISA, it can be used to detect either an antibody (for example, in diagnosing HIV, it tests for the presence of antibodies to the virus), or a target compound (antigen), for example PAPP-A. While this technique is commonly used in immunology, developments in antibody production allow its use in the detection and analysis of a wide range of chemical compounds. It is often the method of choice for detecting biological molecules such as proteins.
ELISA is a test that can be used to detect either antibody (Ab) or antigen such as viral proteins. There are four kinds of ELISA assay tests: Direct ELISA, Indirect ELISA, Sandwich ELISA and Competitive ELISA.
Direct vs Indirect ELISA
- Indirect ELISA: Essentially the method described above.
- Direct ELISA: This is similar to the process described above, except that the primary antibody is linked to the detection enzyme. Therefore, no secondary antibody is required. This method is very quick, but is typically not as sensitive or flexible as the indirect method.
- A less-common variant of this technique, called “sandwich” ELISA, is used to detect sample antigen.
- A soild support is coated with a capture antibody. A solution containing the antigen is added followed by washing. Detection and quantitation of bound antigen is then accomplished by the direct or indirect methodology as above.
- The steps for this ELISA are somewhat different.
- The antigen is bound to the solid support. Unlabeled antibody is incubated in a solution containing the antigen. These bound antibody/antigen complexes are then added to an antigen coated well followed by extensive washes to remove unbound complexes. (The more antigen in the solution, the less antibody will be available to bind to the antigen in the well, hence “competition.”). This is then processed similar to the above methods.
- For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.
Steps in Elisa Testing
Generally, an ELISA has the following steps (each separated by extensive washes):
Antigen Binding: A solid support surface is initially coated with an appropriate amount of the antigen of interest. The whole surface must be blocked to ensure biologically specific binding of the subsequent components. Having the antigen bound to the solid surfaced makes it simple to separate bound from unbound molecules. Non specifically bound materials are washed away during the process of the assay.
Blocking: All unbound sites on the solid support are blocked to prevent nonspecific binding of the antibodies.
Primary Antibody: The primary antibody is added and will be bound if there is a recognized epitope within sample antigen.
Secondary Antibody: An enzyme-linked secondary antibody is added which will bind to any available primary antibody. Primary antibody will only be available on the surface if it has bound to the antigen.
Detection: After adding an enzyme substrate, a colored product will develop if this binding has occurred. The color intensity reflects the amount of primary antibody bound to the target antigen and can be measured.
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