Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is a widely-used method for measuring the concentration of a particular molecule (e.g., a hormone or drug) in a fluid such as serum or urine. It is also known as enzyme immunoassay or EIA.

The molecule is detected by antibodies that have been made against it; that is, for which it is the antigen. Monoclonal antibodies are often used.

The test requires:

  • the antibodies fixed to a solid surface, such as the inner surface of a test tube;
  • a preparation of the same antibodies coupled to an enzyme — one (e.g., β-galactosidase) that produces a colored product from a colorless substrate.

Performing the Test

  1. The tubes are filled with the antigen solution (e.g., urine) to be assayed. Any antigen molecules present bind to the immobilized antibody molecules.
  2. The antibody-enzyme conjugate is added to the reaction mixture. The antibody part of the conjugate binds to any antigen molecules that were bound previously, creating an antibody-antigen-antibody "sandwich".
  3. After washing away any unbound conjugate, the substrate solution is added.
  4. After a set interval, the reaction is stopped (e.g., by adding 1 M NaOH) and the concentration of colored product formed is measured in a spectrophotometer. The intensity of color is proportional to the concentration of bound antigen.

ELISA can also be adapted to measure the concentration of antibodies. In this case,

  1. The wells are coated with the appropriate antigen.
  2. The solution (e.g., serum) containing antibodies is added.
  3. After they have had time to bind to the immobilized antigen,
  4. an enzyme-conjugated anti-immunoglobulin is added, consisting of
    • an antibody against the antibodies being tested for. For example, if human anti-HIV antibodies are being assayed, then antibodies (raised in a goat or rabbit against human immunoglobulins) are conjugated to
    • the enzyme.
  5. After washing away unreacted reagent, the substrate is added.
  6. The intensity of the color produced is proportional to the amount of enzyme-labeled antibodies bound (and thus to the concentration of the antibodies being assayed).

Literally hundreds of ELISA kits are manufactured for

  • research
  • human and veterinary diagnosis

Some examples:

  • screening donated blood for evidence of viral contamination by
    • HIV-1 and HIV-2 (presence of anti-HIV antibodies)
    • hepatitis C (presence of antibodies)
    • hepatitis B (testing for both antibodies and a viral antigen)
    • HTLV-1 and -2 (presence of antibodies)
  • measuring hormone levels
    • HCG (as a test for pregnancy)
    • LH (determining the time of ovulation)
    • TSH, T3 and T4 (for thyroid function)
    • hormones (e.g., anabolic steroids, HGH) that may have been used illicitly by athletes
  • detecting infections
  • detecting allergens in food and house dust
  • measuring "rheumatoid factors" and other autoantibodies in autoimmune diseases like systemic lupus erythematosus
  • measuring toxins in contaminated food
  • detecting illicit drugs, e.g.,
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7 June 2017