1. Radioimmunoassay

2. Radioimmunoassay (RIA) involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantitation using radioactivity.

3. The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g.,
blood banking
diagnosis of allergies
endocrinology

4. 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.

5. Principle
Based on competition between unlabelled antigen and finite amount of corresponding labelled antigen for a limited number of antibody binding sites in a fixed amount of antiserum.
At equilibrium in the presence of an antigen excess there will be both free antigen antigen bound to antibody.

6. Under standard conditions the amount of labelled antigen bound to the antibody will decrease as the amount of unlabelled antigen in the sample increases.

7. 4Ag⃰ + 4 Ab → 4Ag⃰.Ab
4 Ag + 4 Ag⃰ + 4 Ab → 2Ag⃰Ab + 2 AgAb + 2Ag⃰ + 2Ab
12 Ag + 4 Ag⃰ + 4 Ab → Ag⃰ Ab + 3AgAb + 3Ag⃰ + 9 Ag

8. Principle: Uses an immune reaction
[Antigen – Antibody reaction] to estimate a ligand
Ag + Ag* + Ab  AgAb + Ag*Ab + Ag + Ab*
Unbound Ag* and Ag washed out
Radioactivity of bound residue measured
Ligand conc is inversely related to radioactivity
[Ag : ligand to be measured ; Ag* radiolabelled ligand]

9. 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.

11. Gamma Counter

12. From these data, a standard binding curve, like the one shown in red, can be drawn.

13. The samples to be assayed (the unknowns) are run in parallel.
After determining the ratio of bound to free antigen in each unknown, the antigen concentrations can be read directly from the standard curve.

14. The main drawbacks to radioimmunoassay are the expense and hazards if preparing and handling the radioactive antigen.
Both 125I or 131I emit gamma radiation that requires special counting equipment;
The body concentrates iodine atoms — radioactive or not — in the thyroid gland where they are incorporated in thyroxine (T4).

15. anti-DNA antibodies in systemic lupus erythematosus (SLE).
Despite these drawbacks, RIA has become a major tool in the clinical laboratory where it is used to assay
plasma levels of:
most of our hormones;
digitoxin or digoxin in patients receiving these drugs;
certain abused drugs
for the presence of hepatitis B surface antigen (HBsAg) in donated blood;
anti-DNA antibodies in systemic lupus erythematosus (SLE).