1. Radio immunoassay Date – 12.12.2012. Time – 3.30 – 4.30
Dr. Jeevan Shetty MBBS MD
Associate Professor
Reference Book:
Text book of Biochemistry By DM Vasudevan 5th edition

2. At the end of the assignment students should be able to
Definition
Principle of RIA technique
Instrumentation in RIA
Procedure of measurement
Interpretation of results
Advantages and disadvantages of RIA
Application of RIA in medicine and pharmacy

3. Definition - Radioimmunoassay (RIA) involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantitation using radioactivity.
The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma.
It was shown first time that hormone levels in the blood could be detected by an in vitro assay.

4. Radio Immuno Assay (RIA )
Principle
Based on the competition between Unlabeled Antigen (Ag ) and Labeled Antigen (Ag ) for a limited number of antibodies ( Ab )
4Ag+4Ab Ag Ab
4Ag+4Ag+4Ab AgAb+ 2AgAb+2Ag+2Ag
4Ag+12Ag+4Ab AgAb+3AgAb+3Ag+9Ag

5. Under standard conditions, the amount of AgAb (labeled antigen and antibody complex) in the serum will decrease as the amount of unlabeled antigen (Ag in serum )increases
As unlabeled Ag in the serum ↑ AgAb ↓ (Exponentially)
Radioactivity of AgAb is measured
A calibration curve is constructed with different standards

7. Gamma Counter – used for measurement of radioactivity

8. RIA Calibration Curve
From these data, a standard binding curve, like the one shown in red, can be drawn.
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.

9. Applications Estimation of
Hormones - T4 , T3 , insulin, Cortisol, Renin, Aldosterone
Tumor Markers – PSA, AFP
Vitamins
Drugs – Digoxin, phenobarbitone, morphine
For the presence of hepatitis B surface antigen (HBsAg) in donated blood;
Anti-DNA antibodies in systemic lupus erythematosus (SLE).

10. Advantages
Radioimmunoassay is widely-used because of its great sensitivity.
Using antibodies of high affinity, it is possible to detect a few picograms (10−12 g) of antigen in the tube.
The greater the specificity of the antiserum, the greater the specificity of the assay

11. Disadvantages
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).

12. Disadvantages High Cost Short Shelf Life of reagents
Radiological Hazards
Long duration Assay
Problem in disposal