Radioimmunoassay (RIA) requires specialized equipments. It is required to take all precautions which required to handle radioactive material. Hence ELISA is substituted with RIA, In ELISA antigen antibody reactions are estimated by means of colorimetric endpoint indicators in place of a radioactive signal in RIA.
In ELISA enzyme peroxidase reacts with substrate tetra 3’3,5’5 methylebenzidine resulting in change in colour as a indicator for presence of a antigens or antibody.
1.To carry out a radioimmunoassay, a known quantity of an antigen is converted in to a radioactive antigen by tagging it with a gamma-radioactive isotopes of iodine attached to tyrosine ,it is called as radioactive labeled antigen,
2.This radioactive labeled antigen is mixed with a known amount of antibody for that particular antigen,they form radiolabeled antigen (hot antigen) and antibody complex as a result of antigen antibody reaction.
3.Sample of serum (cold antigen) containing unknown quantity to be tested or quantified for a same antigen of that same antigen is added to radiolabeled antigen and antibody complex .
4.A further reaction occurs in between (hot antigen) and (cold antigen) antigen from patients serum, for binding previously added known amount of antibody , the reaction is a competitive between with the radiolabeled antigen and serum antigen for antibody binding sites.
5.As the concentration of serum antigen(cold antigen) increases, the concentration of (cold antigen) binding to the antibody goes on increasing, resulting in the displacement of radiolabeled (hot antigen), At the end point bound antigens are then separated away from the unbound ones, and the radioactivity of the these unbound antigen remaining in the supernatant is estimated.
Radio immunoassay was developed by Rosalyn Yalow and Solomon Aaron Berson , they were awarded with noble prize for sucesfull development of the RIA for quantification of insulin.
Now days RIA is subsituted with ELISA as it is not costly compared to RIA
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