02-02-2013, 12:40 PM
Radioimmunoassay
Radioimmunoassay.ppt (Size: 180 KB / Downloads: 45)
INTRODUCTION
Radioimmunoassay (RIA) involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantitation using radioactivity
The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g.,
blood banking
diagnosis of allergies
endocrinology
The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma.
It represented the first time that hormone levels in the blood could be detected by an in vitro assay.
The Technique
A mixture is prepared of
radioactive antigen
Because of the ease with which iodine atoms can be introduced into tyrosine residues in a protein, the radioactive isotopes 125I or 131I are often used.
antibodies against that antigen.
Known amounts of unlabeled ("cold") antigen are added to samples of the mixture. These compete for the binding sites of the antibodies.
At increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced from the antibody molecules.
The antibody-bound antigen is separated from the free antigen in the supernatant fluid, and
the radioactivity of each is measured.
Performing the Test
The tubes are filled with the antigen solution (e.g., urine) to be assayed. Any antigen molecules present bind to the immobilized antibody molecules.
The antibody-enzyme conjugate is added to the reaction mixture. The antibody part of the conjugate binds to any antigen molecules that were bound previously, creating an antibody-antigen-antibody "sandwich".
After washing away any unbound conjugate, the substrate solution is added.
After a set interval, the reaction is stopped (e.g., by adding 1 N NaOH) and the concentration of colored product formed is measured in a spectrophotometer. The intensity of color is proportional to the concentration of bound antigen.