From WikiVet English
Jump to navigation Jump to search


The radioimmunoassay (RIA) is a sensitive technique used to detect the presence of antigen in a sample using radiolabelled antibodies. Developed in the 1960's, the RIA proved a powerful tool in antigen detection, although the procedure was soon overtaken by ELISA, which utilises enzymes rather than radioactive labels. RIAs are still used today however, to measure:

  • Hormone levels in blood and tissue fluids
  • Serum proteins
  • Drugs
  • Vitamins
    • Levels can be detected when as low as 0.001 micrograms per millilitre, therefore this technique can be use to detect trace amounts of drug


  • The general priniciple behind the RIA is the competitive binding of a radiolabeled antigen and unlabeled antigen to a high-affinity antibody.
  • Labeled antigen is mixed with antibody until binding sites are saturated
  • Samples of unlabeled antigen (unknown concentration) are added in progressively increasing amounts
    • The two different antigens compete for the binding sites on the antibody
    • As the concentration of unlabeled antibody increases, more labeled antigen will be displaced
  • The decrease in bound-radiolabeled antigen is measured - an indication of the amount of antigen present in sample


  • The antigen is often labeled with a gamma-emitting isotope, e.g. iodine-125
    • Beta-emitting isotopes such as tritium are also often used
  1. Determine the amount of antibody required to bind ~50% of radiolabeled antigen in mixture- required to ensure the number of epitopes presented by labeled antigen exceeds number of antibody binding sites
  2. Add unlabeled antigen to mixture
  3. Separate antigen-antibody complex from free antigen by precipitation
  4. Measure radioactivity in precipitate
  • There are various methods to separate the antigen-antibody complexes from the free antigen:
    • Precipitate complexes using secondary isotype-specific anti-immunoglobulin
    • If complex contains IgG, it can be removed by mixing with formalin-killed Staphylococcus aureus- protein A of S. aureus has a high affinity for IgG
      • Removal of the complex by either of these methods leaves an amount of free labeled antigen in the supernatant (liquid section from precipitation)- the radioactivity of this can be measured and the value taken away from the total amount of labeled antigen added (known amount)= amount of bound labeled antigen
    • A number of solid-phase RIAs have been developed
      • Sometimes the antibody can be absorbed onto the surface- the amount of radiolabeled antigen bound to the beads can be measured after washing
      • Antibody can be immobilised on PVC or polystyrene


  • As the technique requires small amounts of sample it can be conducted on 96-well microtiter plates and large numbers of samples can be tested
    • RIA used in this way has been employed in the detection of the hepatitis B surface antigen in donor blood, reducing the occurrence of hepatitis infections as a result of blood transfusions in humans
  • Measuring plasma levels of hormones and controlled substances
  • Measuring anti-DNA antibodies present in systemic lupus erythematosus (SLE)


Although the RIA is a very sensitive test, and therefore widely-used, there are disadvantages to its use:

  • The substances being used are radioactive
  • Gamma radiation needs special counting equipment for detection
  • Iodine is naturally concentrated in the thyroid gland, whether radioactive or not, and incorporated into thyroxine

Consequently, ELISA has largely overtaken RIA as a preferred diagnostic tool

LIVE logo Originally funded by the RVC Jim Bee Award 2007