A technique for determining antibody levels by introducing an antigen labelled with a radioisotope and measuring the subsequent radioactivity of the antibody component.

1. RADIO IMMUNO ASSAY

2. a technique for determining antibody levels by introducing
an antigen labelled with a radioisotope and measuring the
subsequent radioactivity of the antibody component.

3. INTRODUCTION
•RIA is a very sensitive in vitro assay technique used to
measure concentrations of substances, usually
measuring antigen concentrations (for
example, hormone levels in blood) by use of antibodies.
• The technique was first developed in 1960 by two
endocrinologists, S. A. Berson and Rosalyn Yalow, to
determine levels of insulin–anti-insulin complexes in
diabetics.
• In 1977, some years after Berson’s death, the
significance of the technique was acknowledged by the
award of a Nobel Prize to Yalow.

4. PRINCIPLE
• The principle of RIA involves competitive
binding of radiolabeled antigen and unlabeled
antigen to a high-affinity antibody.
• The labeled antigen is mixed with antibody at a
concentration that saturates the antigen-binding
sites of the antibody.
• Then test samples of unlabeled antigen of
unknown concentration are added in
progressively larger amounts.

5. • The antibody does not distinguish labeled from
unlabeled antigen, so the two kinds of antigen
compete for available binding sites on the
antibody.
• As the concentration of unlabeled antigen
increases, more labeled antigen will be displaced
from the binding sites.
• The decrease in the amount of radiolabeled
antigen bound to specific antibody in the
presence of the test sample is measured in order
to determine the amount of antigen present in
the test sample.

6. The antigen is generally labeled with a
gamma emitting isotope such as 125I, buT
beta-emitting isotopes such as tritium (3H) and 14C
are also routinely used as labels.

7. REAGENTS USED IN RID
1. A tracer i.e. a labelled ligand.
2. A binder (Antibody) which is the specific antiserum.
3. A separation system to separate the ‘bound’ and
‘free’ phases.
4. A standard (in highly pure form) .
5. A free human antiserum.

8. PROCEDURE

10. 1. A known quantity of an antigen is made
radioactive .
2. This radiolabeled antigen is then mixed with a
known amount of antibody for that antigen, and
as a result, the two chemically bind to one
another.
3. a sample of serum from a patient containing an
unknown quantity of that same antigen is
added.
4. This causes the unlabeled (or "cold") antigen
from the serum to compete with the
radiolabeled antigen for antibody binding sites .
5. As the concentration of "cold" antigen is
increase, more of it binds to the antibody.

11. 6. By displacing the radio labelled variant ,
reduces the ratio of antibody-bound radio
labelled antigen to free radio labelled antigen.
7. The bound antigens are then separated from
the unbound ones .
8. the radioactivity of the free antigen remaining
in the supernatant is measured.
• HERE separating bound from unbound antigen
is crucial .

12. ADVANTAGES
• Radioimmunoassay is widely used because of
its great sensitivity.
• Using antibodies of high affinity, it is possible
to detect a few picograms of hormone to the
tube.
• The greater the specificity of the antiserum,
the greater the specificity of the assay.

13. DISADVANTAGES
•Prolonged reaction time (in days) as a
consequence highly diluted reagent is used.
•Radioactive Iodine used in is not a cheap
reagent.
•Possible health hazards due to handling of
radioisotopes.
•All the reagents must be added precisely.
•Limited assay range.
•Difficulty of automation.
•Lengthy counting time.

14. USES OF RIA
• Narcotics (drug) detection,
• Blood bank screening for the hepatitis (a
highly contagious condition) virus,
• Early cancer detection,
• Measurement of growth hormone levels,
• Tracking of the leukemia virus,
• Diagnosis and treatment of peptic ulcers, and
• Research with brain chemicals called
neurotransmitters.