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. 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
than antibodies.
4.
5. • 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.
6.
7. 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.
◦ The animal is bled and the antibodies are
collected.
8. 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. Radioimmunoassay (RIA) involves the
separation of a protein (from a mixture) using
the specificity of antibody - antigen binding
and quantitation using radioactivity.
10. Radioimmunoas
say (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. 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.
16. 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.
After determining the ratio of
bound to free antigen in each
unknown, the antigen
concentrations can be read
directly from the standard
curve.
17. 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).
18. 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
19. 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).
20. 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)
22. ◦ 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
23. 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.
24. 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
25. 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
26. Analysis of hormones, vitamins,metabolites,
diagnostic markers
◦ Eg. ACTH, FSH, T3, T4, Glucagon, Insulin,
Testosterone, vitamin B12, prostaglandins,
glucocorticoids,
Therapeutic drug monitoring:
◦ Barbiturates, morphine, digoxin,
Diagnostic procedures for detecting infection
◦ HIV, Hepatitis A, B etc
26
27. • 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;
27
28. In Endocrinology
Insulin, HCG, Vasopressin
Detects Endocrine Disorders
Physiology of Endocrine Function
In Pharmacology
Morphine
Detect Drug Abuse or Drug
Poisoning
Study Drug Kinetics
28
29. Narcotic drug detection
tracking of leukemia virus
diagnosis and treatment of peptic ulcers
research with Neurotransmitters
29
30. 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).
31. 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
32. 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
