Enzyme-Linked Immunosorbent Assay ELISA 1Dr. Nikhat Siddiq

An ELISA (enzyme-linked immunosorbent assay) allows for rapid screening and quantification of the presence of an antigen in a sample. Proteins in a sample are adsorbed to an inert surface, usually a 96- well polystyrene plate. The surface is washed with a solution of an inexpensive nonspecific protein (often casein from nonfat dry milk powder) to block proteins introduced in subsequent steps from also adsorbing to these surfaces. The surface is then treated with a solution containing the primary antibody—an antibody against the protein of interest. Unbound antibody is washed away and the surface is treated with a solution containing antibodies against the primary antibody. These secondary antibodies have been linked to an enzyme that catalyzes a reaction that forms a colored product. After unbound secondary antibody is washed away, the substrate of the antibody- linked enzyme is added. Product formation (monitored as color intensity) is proportional to the concentration of the protein of interest in the sample. 2Dr. Nikhat Siddiq

An ELISA to test for the presence of herpes simplex virus (HSV) antibodies in blood samples. Wells were coated with an HSV antigen, to which antibodies against HSV will bind. The second antibody is antihuman IgG linked to horseradish peroxidase. Blood samples with greater amounts of HSV antibody turn brighter yellow. 3Dr. Nikhat Siddiq

ELISA- Principle 4Dr. Nikhat Siddiq

ELISA 5Dr. Nikhat Siddiq

In an immunoblot assay proteins that have been separated by gel electrophoresis are transferred electrophoretically to a nitrocellulose membrane. The membrane is blocked (as described above for ELISA), then treated successively with primary antibody, secondary antibody linked to enzyme, and substrate. A colored precipitate forms only along the band containing the protein of interest. The immunoblot allows the detection of a minor component in a sample and provides an approximation of its molecular weight. 6Dr. Nikhat Siddiq

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