17.6.4: Immunoassays
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Learning Objectives
- Describe how the Enzyme-linked immunosorbent assay (ELISA) can be used to detect and quantitate antigens, antibodies and allergens
Enzyme-linked immunosorbent assay (ELISA) is a method of quantifying an antigen immobilized on a solid surface. ELISA uses a specific antibody with a covalently coupled enzyme. The amount of antibody that binds the antigen is proportional to the amount of antigen present, which is determined by spectrophotometrically measuring the conversion of a clear substance to a colored product by the coupled enzyme.
Several variations of ELISA, seen in, exist but the most commonly used method is the sandwich ELISA. The sandwich assay uses two different antibodies that are reactive with different epitopes on the antigen with a concentration that needs to be determined. A fixed quantity of one antibody is attached to a series of replicate solid supports, such as plastic microtiter multi-well plate. Test solutions containing antigen at an unknown concentration are added to the wells and allowed to bind. Unbound antigen is removed by washing, and a second antibody which is linked to an enzyme is allowed to bind. This second antibody-enzyme complex constitutes the indicator system of the test. The antigen serves as bridge, so the more antigen in the test solution, the more enzyme-linked antibody will bind. The test solution is used in parallel with a series of standard solutions with known concentrations of antigen that serve as control and reference. The results obtained from the standard solutions are used to construct a binding curve of the second antibody as a function of antigen concentration. The concentration of antigens can be inferred from absorbance readings of standard solutions.
Key Points
- ELISA is a quantitative technique that measures serum concentration of antigens, antibodies, and allergens.
- Standard ELISA uses antibody-antigen-antibody trapping principle with the second antibody coupled to an enzyme. If the complex is formed, the enzyme converts a clear solution into a colored one that can be measured with a spectrophotometer.
- ELISA is performed in a muti-well microtiter plate. In addition to the test solution, standard solutions are added with known antigen concentration. These solutions will be used to infer the concentration of the antigen being tested.
Key Terms
- spectrophotometrically : By using spectrophotometry.
- epitope : That part of a biomolecule (such as a protein) that is the target of an immune response.