1. RADIOIMMUNOASSAY
Presented by:
Chakravarthy.R
Y10MPH1203
Guided by:
Dr. Abdul Rehaman
Asst prof M.ph.,PhD.,
PHARMACEUTICS
NIRMALA COLLEGE OF PHARMACY

2.  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
invitro assay.
Dr. Rosalyn Yalow became the first
female to win a Nobel Prize with
her work on the radioimmunoassay
HISTORY

3. ANTIGENS
 An antigen is a substance with the ability to
induce an immunological response. They
typically enter the body from an infection. They
are recognized at their epitopes by B cells or
by the T cell receptor on T cells.
 Proteins or glycoproteins make the best
antigens because they are the best at
stimulating antigen recognition molecules.
Some immunoassays test for antigens, rather

5. ANTIBODIES
• Antibodies are produced by the
B-lymphocytes. They are glycoproteins
belonging to the “immunoglobulin supergene
family” that are produced in response to a
foreign substance in the body.
• Antibodies have a generally common structure,
but have regions that vary among them to
accommodate the specific antigens.

7. PRODUCTION OF
ANTIBODIES
 Polyclonal antibodies are more likely to
produce a false positive because they are less
specific to antigen epitopes and have varying
binding affinities.
◦ A mouse (or rabbit) is immunized by being
injected with an antigen; the antigen
generates an antibody response in the
animal.

8. LABELS IN
IMMUNOASSAYS
 Immunoassays require the use of labeled
materials in order to measure the amount of
antigen or antibody present. A label is a
molecule that will react as part of the assay,
and in doing so produce a signal that can
be measured in the solution. Examples of a
label include a radioactive compound, or an
enzyme that causes a change of color in a
solution or its fluorescence .

9. PRINCIPLE
 Radioimmunoassay (RIA) involves the
separation of a protein (from a
mixture) using the specificity of
antibody - antigen binding and
quantitation using radioactivity.

10.  Radioimmunoass
ay (RIAs) utilize a
radioactive label
(usually 125I, 3H or
14C), which emits
radiation that can
be measured with
a beta or gamma
counter.

11.  The technique of
radioimmunoassay has
revolutionized research and
clinical practice in many areas,
e.g.,
◦ blood banking
◦ diagnosis of allergies
◦ endocrinology

12. THE TECHNIQUE
 A mixture is prepared of
◦ radioactive antigen
 Because of the ease with which iodine atoms
can be introduced into tyrosine residues in a
protein, the radioactive isotopes 125I or 131I
are often used.
◦ antibodies against that antigen.
 Known amounts of unlabeled ("cold")
antigen are added to samples of the
mixture. These compete for the binding
sites of the antibodies.

13.  At increasing concentrations of unlabeled
antigen, an increasing amount of
radioactive antigen is displaced from the
antibody molecules.
 The antibody-bound antigen is separated
from the free antigen in the supernatant
fluid, and the radioactivity of each is
measured.

15. Gamma Counter

16. CALIBRATION CURVE
 From these data, a
standard binding
curve, like thee one
shown in red, can be
drawn.
 The samples to be
assayed (the
unknowns) are run in
parallel.

17.  After determining the ratio of bound to free
antigen in each unknown, the antigen
concentrations can be read directly from the
standard curve.

18. 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).

19. ADVANTAGES
 Radioimmunoassay is widely-used because
of its great sensitivity.
 Using antibodies of high affinity, it is
possible to detect a few pictograms (10−12
g) of antigen in the tube.
 The greater the specificity of the antiserum,
the greater the specificity of the assay

20.  RIA has become a major tool in the clinical
laboratory where it is used to assay .
 plasma levels of:
◦ most of our hormones;
◦ digitoxin or digoxin in patients receiving these
drugs;
◦ certain abused drugs.
 Presence of hepatitis B surface antigen (HBsAg)
in donated blood.
 Anti-DNA antibodies in systemic lupus
erythematosus (SLE).

21.  Screening donated blood for evidence of
viral contamination by
◦ HIV-1 and HIV-2 (presence of anti-HIV antibodies)
◦ Hepatitis C (presence of antibodies)
◦ Hepatitis B (testing for both antibodies and a viral
antigen)
 Measuring hormone levels
◦ HCG (as a test for pregnancy)
APPLICATIONS

22. Negative :
Positive:

23. ◦ LH (Luteinizing hormone , determining the
time of ovulation)
◦ TSH, T3 and T4 (for thyroid function)
◦ Hormones (e.g., anabolic steroids, HGH) that
may have been used illicitly by athletes.
 Detecting infections
◦ sexually-transmitted agents
◦ like HIV, syphilis, and chlamydia
◦ hepatitis B and C
◦ Toxoplasma gondii

24.  Detecting allergens in food and house
dust .
RAST:
The radioallergosorbent test to detect
specific IgE antibodies to suspected or known
allergens . IgE is the antibody associated with
type I allergic response : Pollen (is a fine to
coarse powder containing the
microgamatophytes of seeds)
The amount of radioactivity is proportional to the
serum IgE for the allergen.

25. RAST
rating
IgE level
(KU/L)
COMMENT
0 < 0.35
Absent or undetectable allergen
specific IgE
1 0.35 - 0.69 Low level of allergen specific IgE
2 0.70 - 3.49 Moderate level of allergen specific IgE
3 3.50 - 17.49 High level of allergen specific IgE
4 17.50 - 49.99 Very high level of allergen specific IgE
5 50.0 - 100.00 Very high level of allergen specific IgE
6 > 100.00
Extremely high level of allergen
specific IgE

26.  Measuring "rheumatoid factors" and other
auto antibodies in autoimmune diseases
like lupus erythematosus.
 Measuring toxins in contaminated food
 Detecting illicit drugs, e.g.,
◦ cocaine
◦ opiates
◦ Δ-9-tetrahydrocannabinol, the active
ingredient in marijuana

27. Detection of drug:

28. DRAWBACKS
 The main drawbacks to radioimmunoassay
are the expense and hazards if preparing
and handling the radioactive antigen.
 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).

29. ISOTOPES
ISOTOPE HALF-LIFE USES
Carbon-11 20.3m Brain scans
Chromium-
51
27.8d Blood Volume
determination
Cobalt-57 270d Measuring vitamin B12
uptake
Cobalt-60 5.26y Radiation cancer therapy
Gadolinium-
153
242d Determining bone density
Gallium-67 78.1 Scan for lung tumors
Iodine-131 8.07d Thyroid therapy
Iridium-192 74d Breast cancer therapy

30. Iron-59 45d Detection of anemia
Phosphorou
s-32
14.3d Detection of skin cancer or
eye tumors
Plutonium-
238
86y Power for pacemakers
Slenium-75 120d Pancreas scans
Sodium-24 15.0h Locating obstructions in blood
flow
Technetium
-99
6.0h Imaging of brain, liver, none
marrow, kidney, lung or heart
Thallium-
201
73h Detecting heart problems with
treadmill stress test
Tritium 12.3y Determining total body water
Xenon-133 5.27d Lung imaging

31. REFARENCES
 Textbook of microbiology by Prescott, Harley.
 Practical analytical chemistry by BECKEET.
 http://en.wikipedia.org/wiki/Radioimmunoass
ay
 http://en.wikipedia.org/wiki/RIA
 http://en.wikipedia.org/wiki/RAST_test
 http://www.millipore.com/immunodetection/id
3/radioimmunoassay.

32. Thank“u”