Radio Immunoassay(RIA)

INTRODUCTION
• Radioimmunoassay (RIA) is an in vitro assay that measures the
presence of an antigen with very high sensitivity.
• Usually presence of any antigen and antibody can be determined and
measured even in minute concentration.
• RIA is one of the most sensitive & specific methods of immune assays
available and the first technique to analyze upto picomolar (1
picomolar = 10-12 molar.)concentration of any biologically
substances.
It involves a combination of three principles.
1. An immune reaction i.e. antigen, antibody binding.
2. A competitive binding or competitive displacement reaction. (It
gives specificity)
3. Measurement of radio emission. (It gives sensitivity)
 1. Immune Reaction
• When a foreign biological substance enters the body’s bloodstream
through a non-oral route, the body recognizes the specific chemistry on
the surface of the foreign substance as antigen and produces specific
antibodies against the antigen so as nullify the effects and keep the body
safe.
• The antibodies are produced by the body’s immune system so, it is an
immune reaction.
 2. Competitive binding or competitive
displacement reaction
• This is a phenomenon wherein when there are two antigens that can
bind to the same antibody, the antigen with more concentration
binds extensively with the limited antibody displacing others. So here
in the experiment, a radiolabelled antigen is allowed to bind to a
high-affinity antibody.
• Then when the patient serum is added to unlabeled antigens it starts
binding to the antibody displacing the labeled antigen.
 3.Measurement of radio emission
• Once the incubation is over, then washings are done to remove any
unbound antigens. Then radio emission of the antigen-antibody
complex is taken, and the gamma rays from the radiolabeled antigen
are measured.
 Radioimmunoassay (RIA) Procedure
1. Specific antibodies of known concentration are fixed in the microtitre well.
2. A known amount of Radiolabelled antigens is then added to the well
3. Washed carefully to remove any unbound antigens
4. At this point, the radioactivity of the well will be maximum.
5. Unlabeled antigens are then added to the well
6. Then these unlabelled antigens will compete with labeled antigen AND it replaces
the labeled antigen by unlabelled antigen.
7. there will be free labeled antigens in the well.
8. Again washed carefully to remove the free labeled antigens.
9. Centrifuge the solution and use the supernatant for the analysis of radioactivity.
10. Radioactivity of wells is then measured by gamma-counter.
Requirements
1.Specific antibodies of known concentration are fixed in the microtitre well.

2.A known amount of Radiolabelled antigens is then added to the well

3. Unlabeled antigens are then added to the well
4. Washed carefully to remove any unbound antigens
5. Decrease in Radioactivity
 6. A standard curve is constructed by plotting the percentage of antibody-
bound radiolabeled antigen against known concentrations of a standardized
unlabeled antigen
7. Determining the concentration of the antigen.
 Radioimmunoassay (RIA) Result
Interpretation
• At first, the labeled antigens will bind to the antibodies hence radioactivity will be maximum.

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

• So by observation of decreasing radioactivity, it can be confirmed that the antigen of interest

is present in the sample. And if the radioactivity remains the same, it can be called a
negative test.

• With the increasing concentration of unlabeled antigens, the radioactivity decreases. By

plotting a graph of radioactivity(in percentage) vs concentration of unlabeled antigens, a
standard curve is obtained.

• The sample to be assayed is run parallel following a similar procedure and the radioactivity
measured is calibrated with the standard curve to determine the concentration of the
antigen
 Radioimmunoassay (RIA) Applications
1. It was first used for the detection of peptide hormones.
2. Detection of different viral antigens
3. Detection of many hormones and drugs
4. Detection of Hepatitis B surface antigens
5. Detection of mycotoxins
6. Detection of the early stage of cancer
 Radioimmunoassay (RIA) Advantages
• High specificity
• High sensitivity
• Can detect a very small amount (nanograms) of antigen or antibodies.
 Radioimmunoassay (RIA) Limitations
• Working with radioactive substances makes it a bit risky.
• Disposal of radioactive substances can be problematic.
• Equipment and reagents are expensive.
• Radiolabeled substances used have a short shelf-life.