Radioimmunoassay: Principle, Uses, Limitations

Radioimmunoassay (RIA) is very sensitive (can detect at a concentration of <0.01 μg/mL) and a specific technique that is used for the quantitative detection of antigens or haptens.

RIA is an extremely important tool in biomedical research and clinical practice. It is commonly used to assay microbial antigens, proteins, vitamins, hormones, or drugs in serum. A specialized RIA called radioallergosorbent test (RAST) is used to measure the amount of serum IgE antibody that reacts with a known allergen (antigen).

Because of the radio hazard associated, the use of RIA is reduced and has been largely replaced by ELISA testing.

Radioimmunoassay was developed by Berson and Yalow (1960), for which they were awarded Nobel prize in 1977.

The principle of RIA is similar to that of competitive ELISA for antigen detection, except that nucleotides (usually ¹²⁵I or ¹⁴C) are substituted for enzymes in RIA.

Principle of Radioimmunoassay (RIA)

Radioimmunoassay uses radioisotope-labeled purified known antigen which competes with unlabeled (unknown) antigen for binding sites on a known amount of antibody.

The Ag-Ab complexes that form between the antigen and antibody can then be precipitated using the second antibody and the amount of radioactivity of the bound complex is measured by means of radioisotope analyzers and autoradiography.

When the sample contains a high amount of antigen, much of the antigen-binding sites of the antibodies are occupied by unlabelled antigen. So the bound complex will show little radioactivity. The radioactivity generated, in fact, is inversely proportional to the amount of antigen present in the sample.

Then the concentration of the unknown (unlabeled) antigen or hapten present in a sample is determined by plotting the value of radioactivity generated in a standard chart generated using different concentrations of same antigens against the radioactivity of the bound complex.

Materials Required for Radioimmunoassay

The materials used in RIA are as follows:

1. Radioactive Isotope: A radioisotope is used as a tracer to label one of the components involved in the assay. Common radioisotopes used include iodine-125 (^125I) and tritium (^3H).
2. Antibodies or Antigens: Specific antibodies or antigens are required depending on the type of assay being conducted. For eg, antibodies specific to a hormone are applicable to measure that hormone. These antibodies or antigens are typically labeled with the radioactive isotope.
3. Sample: The biological sample (e.g., blood, serum, plasma, urine) containing the substance of interest must be collected and prepared for the assay.
4. Standard Curve Standards: A set of standards with known concentrations of the measured substance is prepared. These standards help create a calibration curve, allowing the conversion of radioactive counts into quantitative concentrations.
5. Solid Phase: Tubes or microplates coated with a material that can bind antibodies or antigens are useful to separate bound and unbound components during the assay.
6. Buffer Solutions: Buffers help to create the appropriate conditions for the reaction between antigens and antibodies. They maintain pH and ionic strength to optimize binding.
7. Washing Solution: After the reaction, the washing solution help to remove unbound substances. This increases the assay’s specificity.
8. Gamma Counter: A gamma counter measures the radioactivity in each sample. It quantifies the amount of radioactive tracer present, which is proportional to the concentration of the measured substance.
9. Pipettes and Pipette Tips: Accurate and precise pipetting equipment is essential for preparing standards, samples, and reagents.
10. Safety Equipment: One should wear personal protective equipment, like gloves and lab coats, when handling radioactive materials. Waste disposal procedures must also be proper.
11. Calibrators and Controls: Calibrators with known concentrations and quality control samples are used to ensure the accuracy and reliability of the assay.

Advantages

1. Highly sensitive so it can be used for the detection of small quantities of analyte or antigens.
2. Quantification of antigens or analytes is possible with RIA.

Limitations

1. The handling of radioisotopes requires specific safety measures. Because of this limitation, RIA has been replaced by ELISA in clinical laboratories.

References and further readings

1. Madigan Michael T, Bender, Kelly S, Buckley, Daniel H, Sattley, W. Matthew, & Stahl, David A. (2018). Brock Biology of Microorganisms (15th Edition). Pearson.
2. Willey, Joanne M, Sherwood, Linda M, & Woolverton, Christopher J. (2016). Prescott’s Microbiology (10 edition). McGraw-Hill Education.
3. Tille, P. (2017). Bailey & Scott’s Diagnostic Microbiology (14 edition). Mosby.