ELISA Explained: Principles, Types & Procedures PDF

ELISA Introduction ELISA is the abbreviation for enzyme linked immunosorbent assay. ELISA is a common laboratory technique which is used to measure the concentration of an analyte (usually antibodies or antigens) in solution.  Principle It is based on the principle of antigen-antibody interactio...

ELISA Introduction ELISA is the abbreviation for enzyme linked immunosorbent assay. ELISA is a common laboratory technique which is used to measure the concentration of an analyte (usually antibodies or antigens) in solution.  Principle It is based on the principle of antigen-antibody interaction. Antigen is recognized by specific antibody. To this antigen antibody complex the enzyme attaches which reacts with the substrate to produce product, usually colored. The intensity of color develop is measured at specific nano meter by the ELISA reader which gives the concentration of the analyte to be measured.  Requirements: Solid phase: Microtiter well coated with specific antigen or antibody, having 8×12 wells. Blocking buffer: Block the remaining protein binding site (Bovine serum albumin). Wash buffer: to remove the unbounded antibody. Enzyme conjugate: the enzyme that attaches irreversible to the antibody (horse-redish peroxidase) Chromogen: a chemical that alters color as a result of enzyme interaction with the substrate (trimethyl benzidine) Stopping solution: stop the action of an enzyme on a substrate (HCL) Procedure:  Competitive ELISA Types: Non-Competitive ELISA 1. Direct ELISA 2. Indirect ELISA 3. Sandwich ELISA Non competitive ELISA Non-competitive ELISA does not rely on the competition between target antigens and reference antigens. Direct ELISA Principle: In a direct ELISA, an antigen or sample is immobilized directly on the plate and a conjugated detection antibody binds to the target protein. Substrate is then added, producing a color. Where the intensity of the color developed is directly proportional to concentration of the analyte in the sample. Procedure: Indirect ELISA: Principle: Indirect ELISA is a technique that uses a two-step process for detection, whereby a primary antibody specific for the antigen binds to the target and a labeled secondary antibody against the host species of the primary antibody binds to the primary antibody for detection. Substrate is then added, producing a color. Where the intensity of the color developed is directly Sandwich ELISA Introduction: The sandwich ELISA quantify antigens between two layers of antibodies (i.e. capture and detection antibody). The antigen to be measured must contain at least two antigenic epitope capable of binding to antibody, since at least two antibodies act in the sandwich. Principle Plate is coated with a capture antibody, where sample is added, and any antigen present binds to capture antibody. Then enzyme-linked detecting antibody is added that binds to the antigen. Substrate is then added, producing a color. Where the intensity of the color developed is directly proportional to concentration of the analyte in the sample. Procedure  Competitive ELISA Introduction: It is also known as inhibition ELISA or competitive immunoassay, competitive ELISA assays measure the concentration of an antigen by detection of signal interference. The sample antigen competes with a reference antigen for  Principle The plate is coated with the specific antibody. The sample is pre-incubated with labeled antibody. After washing, the reference antigen (HRP- labeled antigen) is added. Depending on the amount of antigen in the sample, more or less free antibodies will be available to bind the reference antigen. (This means the more antigen there is in the sample, the less reference antigen will be detected and the weaker the signal). Hence, the intensity of color developed is inversely proportional to the concentration of the analyte in the sample. Procedure: Application of ELISA Presence of antigen or the presence of antibody in a sample can be evaluated. Determination of serum antibody concentrations in a virus test. Used in food industry when detecting potential food allergens. Applied in disease outbreaks- tracking the spread of Instrumentation in Biochemistry ELISA washer ELISA Reader Summary Principle: Specific antigen-antibody interaction Types: Competitive ELISA Non-Competitive ELISA 1. Direct ELISA 2. Indirect ELISA 3. Sandwich ELISA