{"id":734,"date":"2022-05-17T18:09:00","date_gmt":"2022-05-17T12:24:00","guid":{"rendered":"http:\/\/immunologynotes.com\/?p=73"},"modified":"2023-08-03T00:17:37","modified_gmt":"2023-08-02T18:32:37","slug":"enzyme-linked-immunosorbent-assay-elisa","status":"publish","type":"post","link":"https:\/\/microbenotes.com\/enzyme-linked-immunosorbent-assay-elisa\/","title":{"rendered":"ELISA- Definition, Principle, Procedure, Types, Steps, Uses"},"content":{"rendered":"\n

Enzyme-Linked Immunosorbent Assay (ELISA) is a modern molecular technique for the detection of antigen-antibody interaction<\/a> with the help of an enzyme.<\/strong><\/p>\n\n\n\n

It is one of the sensitive enzyme immunoassay techniques for the detection of the presence of antigen<\/a> or antibody<\/a> and quantification as well in the case of clinical diagnosis of many diseases.<\/p>\n\n\n\n

Enzyme-linked immunosorbent assay<\/a> (ELISA) utilizes an enzyme system to show a specific combination of an antigen with its antibody. It 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. The ELISA technique was first conceptualized and developed by Peter Perlmann<\/strong> and Eva<\/strong> Engvall<\/strong> at Stockholm University, Sweden.<\/p>\n\n\n\n

\"ELISA
ELISA<\/figcaption><\/figure>\n\n\n\n

The enzyme<\/span> system of ELISA consists enzyme which is labeled to a specific antibody or antigen and a chromogenic substrate that is added after the antigen-antibody reaction. The substrate is hydrolyzed by the enzyme attached to antigen-antibody complexes. An ELISA test uses components of the immune system (such as IgG or IgM antibodies) and chemicals for the detection of immune responses in the body. The ELISA test involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules). Examples of the uses of an ELISA test include diagnosing infections such as HIV (human immunodeficiency virus) and some allergic diseases like food allergies. ELISA tests are also known as immunosorbent assays.<\/span><\/p>\n\n\n\n

Following the antigen-antibody reaction, chromogenic substrate specific to the enzyme (o- phenyldiamine dihydrochloride for peroxidase, p-nitrophenyl phosphate for alkaline phosphatase<\/strong>, etc.) is added. The substrate is acted upon (usually hydrolyzed<\/span>) by an enzyme attached to the <\/span>antigen-antibody complex to give color change. The color in the reaction can be read visually or The reaction is detected by reading the optical density (estimated colorimetrically) using a microassay plate reader i.e. ELISA reader. Usually, a standard curve based on known concentrations of antigen or antibody is prepared from which the unknown quantities are calculated.<\/span><\/p>\n\n\n\n

The antigen or antibody is coated on a <\/span>solid surface such as in a plastic tube or well of a microtiter plate. Thus, after the antigen and antibody have combined (Antigen-antibody complex formed) they remain firmly attached to a solid surface during subsequent washing stages.<\/span><\/p>\n\n\n\n

Enzyme immunoassays (EIAs) can be used for the <\/span>detection of either antigens or antibodies in serum and other body fluids of the patient. In EIA techniques, antigens or antibodies labeled with enzymes are used. Alkaline phosphatase, Horseradish peroxidase, and \u03b2-galactosidase<\/strong> are the enzymes used in the EIA tests.<\/span><\/p>\n\n\n\n

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