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→Technique
==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