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New page: ==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 pro...
==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 utilise 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
==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
# 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 supernate (liquid section from precipitation)- the radioactivity of this can be measured and the value taken away from the amount of labelled antigen= 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
*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 radioactive substances being used
*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
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 utilise 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
==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
# 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 supernate (liquid section from precipitation)- the radioactivity of this can be measured and the value taken away from the amount of labelled antigen= 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
*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 radioactive substances being used
*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