Difference between revisions of "Radioimmunoassay"

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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...)
 
 
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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
 
*Drugs
 
*Drugs
 
*Vitamins
 
*Vitamins
**Levels can be detected when as low as 0.001 micrograms per millilitre
+
**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==
 
==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 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
+
*** 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
 +
 
==Applications==
 
==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
 
*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
+
**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
 +
 +
 +
{{review}}
 +
<br><br>
 +
{{Jim Bee 2007}}
 +
[[Category:Immunological Testing]]

Latest revision as of 16:53, 17 March 2012

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 labeled with a gamma-emitting isotope, e.g. iodine-125
    • Beta-emitting isotopes such as tritium are also often used
  1. 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
  2. Add unlabeled 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 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
      • 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
      • 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

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





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