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− | |linkpage =Immunology - WikiBlood
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− | |linktext =IMMUNOLOGY
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− | |sublink1 =Immunological testing - WikiBlood
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− | |subtext1 =IMMUNOLOGICAL TESTING
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− | |pagetype =Blood
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| ==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 |
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| ==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 |
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| ==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 |
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− | # 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 |
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| **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)]] |
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− | =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 |
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| + | {{review}} |
| + | <br><br> |
| + | {{Jim Bee 2007}} |
| + | [[Category:Immunological Testing]] |