2. Radioimmunoassay
• Radioimmunoassay (RIA) is a very
sensitive in vitro assay technique used
to measure concentrations of antigens
(for example, hormone levels in the
blood) by use of antibodies.
3. • RIA technique is extremely sensitive and
extremely specific, requiring specialized
equipment, it remains the least
expensive method to perform such tests
4. • The technique was introduced in 1960
by berson and yalow as an assay for
the concentration of insulin in plasma.
• It represented the first time that
hormone levels in the blood could be
detected by an in vitro assay.
5. • The technique of radioimmunoassay
has revolutionized research and clinical
practice in many areas, e.G.,
• Blood banking
• Diagnosis of allergies
• Endocrinology
6. Radioimmunoassay: pros and cons
• PRO: versatility : using the same
principle, almost any biomolecule can
be assayed
• Fast (usually 2 days or less)
• Sensitive (comparable to the most
sensitive bioassays, that is < ng/ml)
• Large capacity : thousands of
samples/day specific (antibody-
dependent)
7. Con:
• Use of radioactivity: hazardous
• Expensive equipment (gamma or beta
• Counter)
8. •Principle :
• The technique is based on the ability of
an unlabelled form of the substance to
inhibit competitively the binding of a
radioactively labelled substance by
specific antibodies.
9. Method
• To perform a radioimmunoassay, a
known quantity of an antigen is made
radioactive, frequently by labeling it
with gamma-radioactive isotopes of
iodine attached to tyrosine (hot).
• This radiolabeled antigen is then mixed
with a known amount of antibody for
that antigen, and as a result, the two
specifically bind to one another
10. • Then, a sample of serum from a patient
containing an unknown quantity of that
same antigen is added.
• This causes the unlabeled (or "cold")
antigen from the serum to compete
with the radiolabeled antigen ("hot") for
antibody binding sites.
11. • As the concentration of "cold" antigen
is increased, more of it binds to the
antibody, displacing the radiolabeled
variant, and reducing the ratio of
antibody-bound radiolabeled antigen
to free radiolabeled antigen.
12. The bound antigens are then separated
from the unbound ones, and the
radioactivity of the free antigen
remaining in the supernatant is
measured using a gamma counter.
Using known standards, a binding curve
can then be generated which allows
the amount of antigen in the patient's
serum to be derived.
13. The principle of RIA
• The amount of Ab per tube is kept
constant, the amount of antigen added
(known or unknown) is the variable
parameter.
• The added antigen will be distributed
between a bound (B) and a free (F)
fraction. This distribution is governed by
the association constant (KA) of the Ab:
Ab + Ag AgAb and K = [AbAg]
/[Ab][Ag]
14. • Conclusion: If total Ab input is kept
constant, the value of B/F is a
measure for the total Ag input
15. • To measure this distribution B-F ,
• A small but constant amount g p of
labeled antigen ("tracer") is added to
the reaction.
• Eventually, there will be a competition
reaction between this small but
constant amount of "tracer" and the
"cold" antigen for a limited amount of
antibody.
16.
17.
18. Requirements for the development
of an RIA
1. Pure antigen : for - standards (μg),
- Tracer production (tens of μg)
- Ab production (hundreds of μg)
19. 2. Tracer : self-made or commercial.
3. Specific, high-affinity antibody : self-
made or commercial.
4. A method to separate bound and
free antigen.
5. (Optional) : A system to extract the
antigen from the sample.
20. Separating Bound from Free Antigen
• Precipitate the antigen-antibody
complexes by adding a "second"
antibody directed against the first. For
example, if a rabbit igg is used to bind
the antigen, the complex can be
precipitated by adding an antirabbit-
igg antiserum (e.G., Raised by
immunizing a goat with rabbit igg). This
is the method shown in the diagram
above.
21. • The antigen-specific antibodies can be
coupled to the inner walls of a test tube
After incubation,
• The contents ("free") are removed
• The tube is washed ("bound"),
• The radioactive of both is measured.
• The antigen-specific antibodies can be
coupled to particles, like sephadex.
Centrifugation of the reaction mixture
separates the bound counts (in the pellet)
from the free counts in the supernatant
fluid.
25. • Both 125I or 131I emit gamma radiation
that requires special counting
equipment;
• The body concentrates iodine atoms —
radioactive or not — in the thyroid
gland where they are incorporated in
thyroxine (T4).
26. RIA as a major clinical tool
It is used to assay plasma levels of most
of our hormones
• Digitoxin or digoxin in patients receiving
these drugs
• Certain abused drugs
• For the presence of hepatitis B surface
antigen (hbsag) in donated blood
• Anti-dna antibodies in systemic lupus
erythematosus (SLE).
27. • 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 research with brain chemicals
called neurotransmitters.
