Radioimmunoassay
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Introduction
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
Principle
- 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
Technique
- The antigen is often labelled with a gamma-emitting isotope, e.g. iodine-125
- Beta-emtting isotopes such as tritium are also often used
- Determine the amount of antibody required to bind ~50% of radiolabelled antigen in mixture- required to ensure the number of epitopes presented by labelled antigen exceeds number of antibody binding sites
- Add unlabelled antigen to mixture
- Separate antigen-antibody complex from free antigen by precipitation
- Measure radioactivity in precipitate
- There are various methods to separate the antigen-antibody complexes from the free antigen:
- Precipitate complexes using secondary isotype-specific antiimmunoglobulin
- 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 labelled 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 labelled antigen added (known amount)= amount of bound labelled antigen
- A number of solid-phase RIAs have been developed
- Sometimes the antibody can be absorbed onto the surface- the amount of radiolabelled antigen bound to the beads can be measured after washing
- Antibody can be immobilised on PVC or polystyrene
Applications
- 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)
Drawbacks
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