RIA - Radioimmunoassay

This sensitive immunoassay technique helps in the determination of concentrations of antigens or antibodies in a sample using radioisotopes.

Material Required:

• Microtiter plate and test tubes

• Radiolabelled antigens

• Antibodies

• Unlabelled antigens (sample antigens) - Also called cold antigens

• Washing buffer (1% Trifluoroacetic acid)

• Radioactive counter

There are two counter types used:

1. Gamma counter: Used for gamma energy emitting isotopes.

2. Scintillation counter: Used for beta energy emitting isotopes.

How it works:

1. Buffer is added to wells/ test tubes.

2. A known concentration of the antigen is made radioactive through frequent labelling with gamma-radioactive isotopes.

3. The radioactive antigen is mixed with an antibody with a known concentration, resulting in the antigen and antibody binding together.

4. Wash the plate to remove unbound antigens.

5. An unlabelled antigen/ unknown sample is added.

6. The unknown antigen competes with the radiolabelled antigen for binding sites on the antibody.

7. The radioactive antigens are displaced from the antibody by the unlabelled/ unknown antigen.

8. The plate is washed to remove free labelled antigens.

9. Radioactivity of the wells is measured by a radioactive counter.

Result Interpretation:

• Test tube 1 (Blank): Ab + Ag

• Test tube 2 (Calibrator): Ab + Unlabelled Ag + Ag

• Test tube 3 (sample): Ab + Sample Ag + Agg

A blank is confirmed through the radioactivity remaining the same.

If the sample contains specific antigens of interest, it will bind to the antibodies releasing labelled antigens and the radioactivity of the solution will decrease.

Standard Curve:

To determine the concentration of unknown samples a standard curve is plotted.