Changes

==Introduction==

==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:  

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

*Hormone levels in blood and tissue fluids

*Serum proteins

*Serum proteins

==Principle==

==Principle==

*The general priniciple behind the RIA is the competitive binding of a radiolabelled antigen and unlabelled antigen to a high-affinity antibody.

*The general priniciple behind the RIA is the competitive binding of a radiolabeled antigen and unlabeled antigen to a high-affinity antibody.

*Labelled antigen is mixed with antibody until binding sites are saturated

*Labeled antigen is mixed with antibody until binding sites are saturated

*Samples of unlabelled antigen (unknown concentration) are added in progressively increasing amounts  

*Samples of unlabeled antigen (unknown concentration) are added in progressively increasing amounts  

**The two different antigens compete for the binding sites on the antibody

**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

**As the concentration of unlabeled antibody increases, more labeled antigen will be displaced

*The decrease in bound-radiolabelled antigen is measured- an indication of the amount of antigen present in sample

*The decrease in bound-radiolabeled antigen is measured - an indication of the amount of antigen present in sample

==Technique==

==Technique==

*The antigen is often labelled with a gamma-emitting isotope, e.g. iodine-125

*The antigen is often labeled with a gamma-emitting isotope, e.g. iodine-125

**Beta-emtting isotopes such as tritium are also often used

**Beta-emitting 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''

# 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''

# Add unlabelled antigen to mixture

# Add unlabeled antigen to mixture

# Separate antigen-antibody complex from free antigen by precipitation

# Separate antigen-antibody complex from free antigen by precipitation

# Measure radioactivity in precipitate

# Measure radioactivity in precipitate

*There are various methods to separate the antigen-antibody complexes from the free antigen:

*There are various methods to separate the antigen-antibody complexes from the free antigen:

**Precipitate complexes using secondary isotype-specific antiimmunoglobulin

**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

**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

*** 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

**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

***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

***Antibody can be immobilised on PVC or polystyrene

**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

**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 plasma levels of hormones and controlled substances

*Measuring anti-DNA antibodies present in systemic lupus erythematosus (SLE)

*Measuring anti-DNA antibodies present in [[SLE|systemic lupus erythematosus (SLE)]]

=Drawbacks=

==Drawbacks==

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

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

*The radioactive substances being used

*The substances being used are radioactive

*Gamma radiation needs special counting equipment for detection

*Gamma radiation needs special counting equipment for detection

*Iodine is naturally concentrated in the thyroid gland, whether radioactive or not, and incorporated into thyroxine

*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

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