Radioimmunoassay

IMMUNOLOGY IMMUNOLOGICAL TESTING

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 radiolabelled antigen and unlabelled antigen to a high-affinity antibody.
• Labelled antigen is mixed with antibody until binding sites are saturated
• Samples of unlabelled 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 unlabelled antibody increases, more labelled antigen will be displaced
• The decrease in bound-radiolabelled 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

1. 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
2. Add unlabelled 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 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