Radioisotopes, RIA, ELISA
Objectives
To explain the principle of radioactivity and its various types of
decay.
To list the applications of radioisotopes in the field of medicine.
To state the principle of immunoassays
To list the applications of immunoassays in diagnosing diseases.
ATOM
Isotopes
Isotopes are the elements having the same atomic number but
different mass number
Atomic number = number of protons
Mass number = number of protons + neutrons
Eg; 1H hydrogen, 2H Deuterium, 3H Tritium.
Isotopes may be stable or unstable (radioactive).
Radioisotopes are unstable isotopes that undergoes
spontaneous nuclear degradation and emit energy in the form of
radiation to reach a more stable configuration. The process in
which they release particles and energy is known as decay.
Radioactivity is the spontaneous degradation of nucleus and
transmutation of one element to another with emission of rays
or particles.
Isotopes have similar chemical reactions, since all of them
contain same number of electrons.
Types of Radioactive decay
Radionuclides are the elements capable of undergoing
radioactive decay. There are three types of decay
alpha decay
beta decay
gamma decay
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http://www.buzzle.com/images/diagrams/gamma-decay.jpg
Gamma ray has no mass and no charge, therefore penetration
power is maximum.
Hence Gamma rays are widely used for treatment of cancer cases
http://www.frankswebspace.org.uk/ScienceAndMaths/physics/physicsGCE/images/penetration.jp
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Applications of Radioactivity
Research
Diagnostic application
Therapeutic application
Applications of Radioactivity in
Research
Since isotopes of an element have identical chemical reactions, when a
radiolabeled compound is administered, these molecules are
metabolized by the body similar to the normal molecules. This is called
“Tracer technique”.
Almost all the biochemical pathways were studied by using tracers. Eg;
14 C – isotope labeled metabolites
Turnover rate of a substance in the body, that is the rate of synthesis
and breakdown could also be studied using tracer techniques.
Applications of Radioactivity in
Research
The total body content of a particular substance can be
quantitated by isotope dilution technique. Eg; 3H labeled
water to determine total body water.
32 P is used to trace nucleic acid synthesis. 3H-labeled thymidine
is incorporated in newly synthesized DNA and therefore used in
assessing cell division kinetics.
Carbon dating technique is an important tool in paleobiology
Applications of Radioactivity for
Diagnosis
The branch of medicine that deals with the diagnostic
applications of radioactivity is referred to as Nuclear medicine.
Radioactive tracers uses gamma ray emitters and gamma-ray
counters to detect them.
Lifespan of RBC and intravascular hemolysis can be detected by
tagging RBCs with 51 Cr.
Thyroid uptake studies by 131 I are used to detect functional
derangements of thyroid gland. Increased uptake in
hyperthyroidism and decreased uptake in hypothyroidism.
Thyroid scanning after intravenous administration of 131I gives an
idea about the approximate size and shape of thyroid gland. Defective
uptake of iodine in certain circumscribed region of the gland known
as “silent nodule” is suggestive of cancer thyroid
Bone scanning by 90 Sr (radioactive strontium) to detect osteoblastoma.
Kidney scanning by 131 I-labeled hippuran to detect anatomical and
physiological defects.
PET scan with a series of images tells us about the functional status
of an organ. PET scan is widely used in oncology with 18 F as a tracer.
Radioimmunoassays (RIA)
Applications of Radioactivity in
Treatment
131I is used for the treatment of hyperthyroidism(Graves’
disease) and thyroid cancers.
Treatment of cancers (radiotherapy).
Radiotherapy mainly affects cells in the division phase. Since
cancer tissue has more dividing cells than normal tissues, cancer
cells are preferentially affected by radiation.
Adverse effects of radiation on normal
tissues
Skin: Radiodermatitis, leading to erythema, atrophy of skin,
fibrosis and loss of elasticity.
Mucous membrane : Nausea, vomiting, diarrhoea, ulceration and
fibrosis in gastrointestinal mucosa as they are very sensitive to
radiation.
Blood cells : Bone marrow is highly radiosensitive leading to
leukopenia and thrombocytopenia.
Gonads : They are highly radiosensitive and leads to sterility in
high doses.
IMMUNO ASSAYS
The exquisite specificity of antigen-antibody interactions has led
to the development of a variety of immunologic assays, which
can be used to detect the presence of either antibody or
antigen.
Radioimmunoassay (RIA)
Enzyme-Linked Immunosorbent Assay (ELISA)
Dr. Rosalyn Yalow and Dr. Sol Berson
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Radioimmunoassay
The principle of RIA involves competitive binding of
radiolabeled antigen and unlabeled antigen to a high-
affinity antibody.
Step 1:
The labeled antigen is mixed with antibody at a concentration
that saturates the antigen-binding sites of the antibody.
Step 2:
Test samples of unlabeled antigen of unknown concentration
are added.
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.
Step 3:
To determine the amount of labeled antigen bound, the Ag-Ab
complex is precipitated and the radioactivity in the precipitate is
measured.
Step 4:
A standard curve can be generated using unlabeled antigen
samples of known concentration (in place of the test sample),
and from this plot the amount of antigen in the test mixture may
be precisely determined.
Radioimmunoassay
Kuby Immunology 5th edition
Applications of RIA
It is used to measure a wide variety of analytes:
– Hormones
– Tumor markers
– Vitamins
– Cytokines
– Blood levels of drugs.
It is extremely sensitive and very accurate
Disadvantages of RIA
Health hazards associated with radioactive isotopes.
Safe disposal of radioactive waste is a problem.
All laboratories do not have the facilities to use radioisotopes.
Isotopes have a definite half-life (reagents have to be replaced
frequently)
Assay takes a long time (~1 day)
ELISA
Enzyme-linked immunosorbent assay, commonly known as
ELISA, is similar in principle to RIA but depends on an enzyme
rather than a radioactive label.
An enzyme conjugated with an antibody reacts with a colorless
substrate to generate a colored reaction product.
Colored end product correlates to the amount of analyte present in
the original sample.
Commonly used enzymes for ELISA includes
alkaline phosphatase,
horseradish peroxidase
These assays approach the sensitivity of RIAs and have the advantage
of being safer and less costly
Overview of steps in ELISA
Coating:
Polystyrene plate is treated with a solution of
either antigen or antibody
Blocking:
An unrelated protein based solution is used to
cover all unbound sites on the plate
Antibody-antigen interaction:
Enzyme conjugated antibody specifically binds
to target antigen or antibody
Detection:
Substrate specific for the enzyme is added and the
intensity of the color produced by the enzyme substrate reaction
is measured by spectrophotometry
Types of ELISA
ELISA can be used to detect qualitatively or quantitatively
the presence of antibody or antigen.
Types of ELISA:
Direct ELISA
Indirect ELISA
Sandwich ELISA
Direct ELISA
An antigen coated to a multiwell plate is detected
by an antibody that has been directly conjugated
to an enzyme.
Advantage:
It is performed faster since fewer steps are
Involved.
Disadvantage:
Less signal amplification and hence lower sensitivity
Indirect ELISA
Antigen coated to a polystyrene multiwell plate is
detected in two stages
First an unlabeled primary antibody, which is
specific for the antigen, is applied.
Next, an enzyme-labeled secondary antibody
specific to the primary antibody is added.
In indirect ELISA, signal amplification is good and
hence has higher sensitivity.
INDIRECT ELISA
Kuby Immunology 5th edition
SANDWICH ELISA
Antigen can be detected or measured by a
sandwich ELISA
Step 1: The first antibody, termed the capture
antibody is immobilized on a microtiter well.
Step 2: A sample containing antigen is added and
allowed to react with the immobilized antibody.
Step 3: After washing the well, a second enzyme-
linked antibody specific for a different epitope on
the antigen is added and allowed to react with the
bound antigen.
Step 4: Substrate is added, and the colored
reaction product is measured.
SANDWICH ELISA
Kuby Immunology 5th edition
Detection/Quantification
Substrate and the enzyme forms the colored reaction product.
H2 O2 HRP H2 O + Nascent oxygen (o)
Diaminobenzidene Oxidised DAB
(Colorless) (Brown color)
The intensity of the color is measured using a
spectrophotometer.
Chemiluminescence assays are preferred over
chromogenic assay due to the enhanced sensitivity of
chemiluminescence.
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Applications of ELISA
• Detection of HIV antibodies in blood sample.
• Detection of hepatitis B infection.
• Detection of cytokines
REFERENCES
1) Textbook of Biochemistry for medical students.
D.M.Vasudevan. Seventh edition
2) Immunology. Kuby. Fifth edition
3) Tietz, Textbook of clinical chemistry. Fifth edition
4) Biorad, ELISA protocol: https://www.bio-rad-
antibodies.com/elisa-types-direct-indirect-sandwich-
competition-elisa-formats.html
