RIA

Radioimmunoassays
• Radioimmunoassays (RIAs) use antibodies to detect and quantitate
the amount of antigen (analyte) in a sample.
• These assays are typically very sensitive and specific. It is possible
to detect as low as a few picograms of analyte in the experimental
tube when using antibodies of high affinity (Kd = 10-8 - 10-11 M).
• The basic principle of radioimmunoassay is competitive binding,
where a radioactive antigen ("tracer") competes with a non-
radioactive antigen for a fixed number of antibody or receptor
binding sites.
• When unlabeled antigen from standards or samples and a fixed
amount of tracer (labeled antigen) are allowed to react with a
constant and limiting amount of antibody, decreasing amounts of
tracer are bound to the antibody as the amount of unlabeled
antigen is increased.
• What is Radioimmunoassay (RIA)?
• Radioimmunoassay is one of the sensitive immunoassay techniques
which helps in the determination of antigen or antibodies in a
sample with the use of radioisotopes.e
• It is an in vitro type of antigen-antibody interaction.
• Invented in 1960 by Rosalyn Yalow and Solomon Berson at Veterans
Administration Hospital, New York.
• Radioimmunoassay (RIA) Requirements
• Radiolabeled antigens: The antigens are generally labeled with
gamma-ray emitting isotopes such as I-125 and beta-ray emitting
isotopes such as Tritium. They are also called hot antigens.

• Specific Antibodies: They are required in smaller amounts than

antigens.

• Unlabeled antigens (sample antigens): They are also called cold

antigens.

• Microtitre plates: 96 wells microtitre plate

• Washing Buffer solutions: Wash buffer such as 1% Trifluoroacetic

acid is used.
Radioimmunoassay (RIA) Principle
• Antigen and antibodies bind specifically to form the Ag-Ab
complex.
• The antigen can be labeled or conjugated with
radioisotopes.
• The unlabeled antigens from the sample compete with
radiolabeled antigens to bind on paratopes of specific
antibodies. The unlabeled antigens replace labeled antigens
that are already linked with the antibodies.
• The unlabeled antigens when bind with antibodies, increases
the amount of free radiolabeled antigens in the solution.
Hence the concentration of free labeled antigens is directly
proportional with the bound unlabeled antigens.
Radioimmunoassay (RIA) Procedure
• Specific antibodies of known concentration are fixed in the microtitre well.
• A known amount of hot antigens is then added to the well
• Washed carefully to remove any unbound antigens
• At this point, the radioactivity of the well will be maximum.
• Unlabeled antigens are then added to the well
• The unlabeled antigens will bind to the antibodies and there will be free
labeled antigens in the well.
• Again washed carefully to remove the free labeled antigens.
• Radioactivity of wells is then measured by gamma-counter.

Fig- Radioimmunoassay (RIA)

Procedure
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 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
• It was first used for the detection of peptide hormones.
• Detection of different viral antigens
• Detection of many hormones and drugs
• Detection of Hepatitis B surface antigens
• Detection of mycotoxins
• Detection of the early stage of cancer

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.
Fig- Illustrates how a limited amount of antibody binding sites
contained in a test tube or microplate well can either bind
unlabeled ligand (in this example, Hepatitis B Surface Antigen) or
radiolabeled ligand. As the amount of unlabeled ligand increases,
there is consequently less radiolabeled ligand bound. The
unlabeled ligand can come from either a “calibration standard” or
the sample that you are trying to measure.