ELISA
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- For the Finnish telecommunications company, see Elisa Oyj.
- For the artificial intelligence program, see ELIZA
The Enzyme-Linked Immunosorbent Assay (ELISA or EIA for short) is a method usually employed in biochemistry to detect the presence of a certain substance in a sample. It utilizes antibodies specific to the substance (antigen); these antibodies are linked to an enzyme which causes a chromogenic or fluorogenic substrate to produce a signal.
The steps of the general, non-competitive ELISA are:
- Apply the sample to a surface, often the well of a microtiter plate. The plate wells or other surface are coated with the primary detection antibody to bind the antigen in the sample solution.
- Wash the plate, so that unbound antigen is removed. Note: this wash is not performed if the sample and enzyme-linked antibody are co-incubated
- Apply the enzyme-linked antibodies which will bind to the antigen if it is present and captured on the detection antibody
- Wash the plate, so that excess unbound antibodies are removed.
- Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorescent signal.
- View the result using a spectrophotomter or other optical device.
The enzyme acts as an amplifier: even if only few enzyme-linked antibodies are present, the enzyme molecules will produce many fluorescent signal molecules.
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analystand may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve which is typically a serial dilution of the target.
A variant of this technique is used in medicine to detect if a person's blood contains antibodies against a certain antigen (which would indicate past or present infection). The initial screening test for HIV infection is such an ELISA test. The steps are as follows:
- Prepare a plate to which the antigen is bound
- Apply the human serum to be tested
- Wash the plate, so that unbound antibodies are removed.
- Apply the enzyme-linked antibodies which specifically bind to the human antibodies.
- Wash the plate, so that unbound enzyme-linked antibodies are removed.
- Apply a chemical which is converted by the enzyme into a fluorescent signal.
- View the result: if it fluoresces, then the serum sample contained antibodies against the antigen.
See also:
External links
- An animated illustration of an ELISA assay (http://www.immunospot.com/elisa-animation.html)
- An animated tutorial comparing the direct and indirect ELISA methods (http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/ELISA.html)
- Online bookshelf from NCBI: searchable text books in molecular biology and related fields (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books)
- "Introduction to ELISA Activity - beginner walkthrough of ELISA used for detecting HIV, including animations (http://www.biology.arizona.edu/immunology/activities/elisa/main.html)
- Image of a plate, such as is commonly used in ELISA tests (http://www.freewebs.com/immunology/elisa-plate.html)de:Enzyme-linked Immunosorbent Assay