RadioImmunoAssay | The principle and Procedure of RIA
Radioimmune assay (RIA): As the name indicates, it is an immunological assay to analyze any antigen or antibody in the patient’s serum to diagnose the disease.
This is one of the most sensitive & specific methods of immune assays available.
It involves competitive binding of radio-labeled antigen and unlabeled antigen to a high-affinity antibody.
The sensitivity range is 0.0006–0.006 µg antibody/ml.
The technique was developed by S. A. Berson, and Rosalyn Yalow and Rosalyn R. Yalow received the Nobel Prize for it in 1977.
It involves three principles which make it most specific & sensitive than other immune assays.
- An immune reaction, i.e., antigen, antibody binding.
- A competitive binding or competitive displacement reaction. (It gives specificity)
- Measurement of radio emission. (It provides sensitivity)
When a foreign biological substance enters into body bloodstream through non-oral route, the body recognizes the specific chemistry on the surface of the foreign material as antigen and produces specific antibodies against the antigen so as nullify the effects and keep the body safe. Body immune system produces the antibodies so; it is an immune reaction. Here the antibodies or antigens bind move due to chemical influence. This is different to Principle of electrophoresis where proteins are separated due to charge.
A competitive binding or competitive displacement reaction:
This is a phenomenon wherein when there are two antigens which can bind to the same antibody, the antigen with more concentration binds extensively with the limited antibody displacing other. So here in the experiment, the radiolabelled antigen is allowed to bind to high-affinity antibody. Then when patient serum is added unlabelled antigens in it start binding to the antibody displacing the labeled antigen.
Measurement of radio emission:
Once the incubation is over, then washings are done to remove any unbound antigens. Then radio emission of the antigen-antibody complex is taken, the gamma rays from radiolabeled antigen are measured.
The approach for RIA:
The first step to set up an RIA is to determine the amount of antibody needed to bind 50–70% of a fixed quantity of radioactive antigen (Ag*) in the assay mixture. This ratio of antibody to Ag* is chosen to ensure that the number of epitopes presented by the labeled antigen always exceeds the total number of antibody binding sites. Consequently, unlabeled antigen (from patient serum) added to the sample mixture will compete with a radiolabeled antigen to bind to the limited number of antibody.
Even a small amount of unlabeled antigen added to the assay mixture of labeled antigen and antibody will cause a decrease in the amount of radioactive antigen bound, and this decrease will be proportional to the amount of unlabeled antigen added. To determine the amount of labeled bound antigen, the Ag-Ab complex is precipitated to separate it from free antigen (antigen not bound to Ab), and the radioactivity in the precipitate is measured. A standard curve can be generated using unlabeled antigen samples of known concentration (in place of the test sample), and from this plot, the amount of antigen in the test mixture may be precisely determined.
Procedure of RIA
- The labeled antigen is mixed with antibody at a concentration that saturates the antigen-binding sites of the antibody.
- Then test samples of an unlabeled antigen of unknown concentration are added in progressively more substantial amounts.
- The antibody does not distinguish labeled from the unlabeled antigen, so the two kinds of antigen compete for available binding sites on the antibody. As the concentration of unlabeled antigen increases, the more labeled antigen will be displaced from the binding sites.
- The decrease in the amount of radiolabeled antigen bound to the specific antibody in the presence of the test sample is measured to determine the amount of antigen present in the test sample.
- The antigen is generally labeled with a gamma-emitting isotope such as I125, but beta-emitting isotopes such as tritium (3H) are also routinely used as labels.
- The radiolabeled antigen is part of the assay mixture; the test sample may be a complex mixture, such as serum or other body fluids, that contains the unlabeled antigen.
The procedure requires small amounts of sample and can be conducted in small 96-well microtiter plates; hence this procedure is suitable to determine the concentration of a particular antigen in large numbers of samples. For example, a microtiter RIA can be used to screen for the presence of the hepatitis B virus. RIA screening of donor blood has sharply reduced the incidence of hepatitis B infections in recipients of blood transfusions
Ref: Kuby Immunology.