The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g.,
- blood banking
- diagnosis of allergies
The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma. It represented the first time that hormone levels in the blood could be detected by an in vitro assay.
- A mixture is prepared of
- radioactive antigen
- Because of the ease with which iodine atoms can be introduced into tyrosine residues in a protein, the radioactive isotopes 125I or 131I are often used.
- antibodies ("First" antibody) against that antigen.
- Known amounts of unlabeled ("cold") antigen are added to samples of the mixture. These compete for the binding sites of the antibodies.
- At increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced from the antibody molecules.
- The antibody-bound antigen is separated (see below) from the free antigen in the supernatant fluid, and
- the radioactivity of each is measured.
- From these data, a standard binding curve, like this one shown in red, can be drawn.
- The samples to be assayed (the unknowns) are run in parallel.
- After determining the ratio of bound to free antigen ("cpm Bound/cpm Free") in each unknown, the antigen concentrations can be read directly from the standard curve (as shown above).
Separating Bound from Free Antigen
There are several ways of doing this.
Radioimmunoassay is widely-used because of its great sensitivity. Using antibodies of high affinity (K0 = 108–1011 M−1), it is possible to detect a few picograms (10−12 g) of antigen in the tube.
- Precipitate the antigen-antibody complexes by adding a "second" antibody directed against the first. For example, if a rabbit IgG is used to bind the antigen, the complex can be precipitated by adding an antirabbit-IgG antiserum (e.g., raised by immunizing a goat with rabbit IgG). This is the method shown in the diagram above.
- The antigen-specific antibodies can be coupled to the inner walls of a test tube [View another example]. After incubation,
- the contents ("free") are removed;
- the tube is washed ("bound"), and
- the radioactive of both is measured.
- The antigen-specific antibodies can be coupled to particles, like Sephadex. Centrifugation of the reaction mixture separates
- the bound counts (in the pellet) from
- the free counts in the supernatant fluid.
The greater the specificity of the antiserum, the greater the specificity of the assay.
The main drawbacks to radioimmunoassay are the expense and hazards of preparing and handling the radioactive antigen.
- Both 125I or 131I emit gamma radiation that requires special counting equipment;
- The body concentrates iodine atoms — radioactive or not — in the thyroid gland where they are incorporated in thyroxine (T4).
|The enzyme-linked immunosorbent assay (ELISA) has many of the advantages (e.g., sensitivity, ease of handling multiple samples) without the disadvantages of dealing with radioactivity. Link to a description of ELISA|
Despite these drawbacks, RIA has become a major tool in the clinical laboratory where it is used to assay
9 March 2011