Radio Immuno Assay (RIA) Immuno Radio Metric Assay (IRMA)

1. Radio Immuno-Assay
(RIA)
Or
Immuno Radio Metric Assay (IRMA)

2. Contents:
• Definition
• History
• Key Terms
• Qualitative Method
• Quantitative Method
• Common Applications
• Relative Comparison

3. In one sentence:
Radioimmunoassay is a laboratory technique that uses
radioactive isotopes to detect and measure very small amounts
of antigens in a sample.

4. Little History:
It was made in New York (1959)
Dr. Yalow won a Nobel Prize for
medicine, the 2nd woman ever to
win it.
Solomon Berson was her partner.

5. Before we move in to the method:
• Hot Antigen
• Radiolabeled antigen (Ag*)
• Cold Antigen
• Un-labeled antigen (Ag)
• Competitive binding
• Due to radioactivity the Ag* loses its binding potency and Ag is able to replace it out.
• 1% TFA buffer
• This helps sediment blood proteins
• Radioactive material
• I125 C14 H3

6. Qualitative method:
1. Coat one well of a microtiter plate with known amount of antibody for the concerned antigen
2. Take known amount of synthetic radiolabeled antigen of the concerned such that it will completely
saturate all the binding sites of antibodies in the well.
3. Wash way the unbound antigens with buffer (1% TFA)
4. Now place the patient sample in the well

7. 1. Through competitive binding the radiolabeled antigens will be replaced by the unlabeled
antigens of the patient sample.
2. Wash away the well with buffer and store the contents in a test tube
3. After centrifugation we assay the supernatant with gamma counter for radioactivity.
4. The amount of radioactivity will be approximately equal to the amount of concerned antigen
present in the patient sample.
5. With presence of radioactivity we conclude that sample contains the concerned antigens

8. Quantitative method:
• Step 1: We will obtain a standard curve to accurately estimate the amount of antigens in a sample.
1. We take 3 components of known quantity : immobilized antibody, radiolabeled antigen, unlabeled antigen.
2. We take 5 microtiter plate for simplicity.
3. We will place say 3 Abs in each well.
4. In all the wells we add enough Ag* such that all of the binding sites are saturated. We make the 1st well our
control.
5. Now we will add increasing amount of Ag in all of the wells except the first. I.e. Ag* (same conc.) + Ag
(increasing conc.)
6. With increasing conc. Ag occupies more and more binding sites.
7. After washing, we will measure the radioactivity of each well.
8. The radioactivity will decrease with increasing known concentration of Ag
9. Now we plot the standard curve where Y- axis (radioactivity %) and X-axis (Ag conc.)

9. Quantitative method:
• Step 2: Now we will obtain the radioactivity of patient sample
1. Taking the same amount of immobilized Ab, Ag* we will add patient serum suspension.
2. After rinsing we measure the radioactivity.
3. Let’s say we obtain 35% of radioactivity
4. We will extrapolate this value on the standard curve and thus we will get the concentration
of Ag
0
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0 2 4 6 8 10 12
Radioactivity
(%)
Ag. Concentration
Standard Curve
0
10
20
30
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0 2 4 6 8 10 12
Radioactivity
(%)
Ag. Concentration
Standard Curve

10. Common applications
• The level of a specific antigen in the blood of the patient.
• Hormone levels detection (e.g. insulin).
• Allergen detection: RAST (Radio Allergen Sorbent Assay)
• Blood bank contaminant detection (e.g. hepatitis virus)

11. Relative comparison:
• Advantages:
• Very specific
• Very sensitive (can work with sample sizes as small as picograms 10-10)
• Drawbacks:
• Radioactive isotopes have very small shelf life
• Time consuming
• Radioactive hazards

12. Questions?