ELISA is a test to determine protein levels in a biological sample. There are two types: indirect ELISA and sandwich ELISA. The ELISA detection step involves adding an enzyme-labeled antibody and a substrate, resulting in a color change. Horseradish peroxidase and alkaline phosphatase are common enzymes used. The selection of enzyme substrate combinations is based on commercially available options and equipment used. ELISA is a versatile tool in disease testing and research.
An enzyme immunosorbent assay (ELISA) is a test performed in an immunology laboratory to determine the levels of proteins in a biological sample. ELISA detection refers to the final step of the test in which a clear solution, or substrate, is added to a plastic plate containing an enzyme-bound labeled antibody. The enzyme breaks down the substrate and a color change occurs. The light absorbance of the final colored solution is then measured on an ELISA plate reader or spectrophotometer.
There are various types of ELISA tests, the two most common being the indirect ELISA and the capture, or sandwich, ELISA. Indirect ELISA is used to detect a protein, known as an antibody, in a patient’s serum. An example of an indirect ELISA test is the human immunodeficiency virus (HIV) test used to detect antibodies to HIV. The ELISA sandwich test detects a protein, or antigen, by capturing it between two antibodies. The detection of human chorionic gonadotropin hormone (hCG), which is elevated during pregnancy, is performed with a sandwich ELISA test.
Both tests have an ELISA detection step at the end of the test. This step involves adding an antibody to which an enzyme molecule is attached. After the addition of the enzyme-labelled antibody, a colorless solution is added, containing the specific substrate molecule for that enzyme. The enzyme breaks down the substrate molecule and the solution changes color depending on the combination used. Determination of the amount of antibody or antigen in the patient sample is done by measuring the intensity of the color change.
Several combinations of enzyme substrates are available for use in the ELISA detection step. The most common enzyme is horseradish peroxidase (HRP), which can cleave the substrate molecules ortho-phenylenediamine dihydrochloride (OPD) and tetramethylbenzidine (TMB), among others. The cleavage of both OPD and TMB results in a yellow color and the optical density or light absorbance of these substrates is measured by an ELISA plate reader. The light absorption of OPD is measured at a wavelength of 490 nanometers (nm) while TMB is measured at 450 nm.
Another common enzyme used in the ELISA detection step is alkaline phosphatase. This enzyme is used with the substrate p-nitrophenyl phosphate (PNPP) and also produces a yellow solution. PNPP absorbs light at a wavelength of 405 nm.
Selection of enzyme substrate combinations is usually based on which enzyme-labeled antibodies are commercially available, as well as what equipment will be used to measure light absorption. The many combinations available for ELISA detection make the ELISA test highly versatile. It is an important tool in disease testing and research laboratories.
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