Ria

RADIOIMMUNOASSAY
1
By
SUNILBOREDDY

IMMUNOASSAYS
“Immuno” refers to an immune response that
causes the body to generate antibodies.
“Assay” refers to a test..
An Immunoassay is a test that uses
immunocomplexing when antibodies and
antigens are brought together.
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Categories of Immunoassay Tests
Competitive
Noncompetitive
Homogeneous
Heterogeneous 3

Labels may be applied to eitherthe
antibody
..orthe antigen.
4

Competitive Assays
• Unlabeled analyte (antigen) in the
test sample is measured by its
ability to compete with the
labeled antigen in the
immunoassay.
• In a competitive immunoassay,
less label measured in the assay
means more of the unlabeled (test
sample) antigen is present. 5

• Noncompetitive assay formats give the highest
level of sensitivity and specificity.
• They are normally used to measure critical
analytes such as cardiac and hepatitis markers.
Noncompetitive Assays
• The measurement of the labeled analyte (Ab)
α amount of Ag present in the sample
6

• There are two versions of the Non
competitive format:
• One step format
Two step format
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Heterogeneous and Homogeneous
immunoassays Methods
• Immunoassays that require separation of the
bound Ab-Ag* complex are referred to as being
HETEROGENEOUS IMMUNOASSAYS.
• Those that do not require separation are
referred to as HOMOGENEOUS IMMUNOASSAYS.
• Homogeneous methods have generally been
applied to the measurement of small analytes
such as Ab used and therapeutic drugs.
8

Radioimmunoassay (RIA)
Enzyme Immunoassay (ELISA)
Fluorescence Polarization Immunoassay
(FPIA)
9

•The technique was introduced in 1960 by Berson
and Yalow as an assay for the concentration of
insulin in plasma.
•Here radioactive materials are not administered
to the individual but are used as reagents.
Radioimmunoassay (RIA)
• Involves the separation of a protein (from a mixture)
using the specificity of antibody - antigen binding and
quantify it using radioactivity
11

The technique of radioimmunoassay has
revolutionized research and clinical practice
in many areas, e.g.,
blood banking
diagnosis of allergies
endocrinology
12

Principle of RadioimmunoassayPrinciple of Radioimmunoassay
Principle:
Uses an immune reaction [Antigen –
Antibody reaction] to estimate a ligand
Ag + Ag* + Ab  AgAb + Ag*Ab + Ag + Ab*
◦ Unbound Ag* and Ag washed out
◦ Radioactivity of bound residue measured
◦ Ligand conc. is inversely related to
radioactivity
[Ag : ligand to be measured ; Ag* radiolabelled
ligand]
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+
+P
P*
P*Q
Radioactive tag
Analyte
Binding agent
Free Bound
Q
PQ
14

A mixture is prepared of
radioactive antigen
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.
15

 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.
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Requirements for RIARequirements for RIA
1. Preparation & characterisation
of the Antigen [Ligand to be
analysed]
2. Radiolabelling of the Antigen
3. Preparation of the Specific
Antibody
4. Development of Assay System
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Preparation & Radiolabelling of the AgPreparation & Radiolabelling of the Ag
Antigens prepared by..
◦ Synthesis of the molecule
◦ Isolation from natural sources
Radiolabelling [Tagging procedure]
◦ 3
H ,14
C, 125
I are used as radioactive tags
◦ Antigens are tagged to 3
H ,14
C, 125
I
◦ Tagging should NOT affect Antigenic
specificity & Antigenic activity !
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Preparation of the Specific AntibodyPreparation of the Specific Antibody
Antigen injected intradermally into
rabbits or guinea pigs  antibody
production
Antibodies recovered from the serum
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Development of the Assay SystemDevelopment of the Assay System
 Crucial step is separation of unbound
antigens
 Antibodies bind to microtitre well
surface [Solid phase RIA]
Antigens bound to the fixed antibodies
remain stuck to the inner surface
Decanting & washing the well removes
unbound antigens
Other techniques of separation:
Centrifugation, Precipitation and
Electrophoresis 22

Assay ProcedureAssay Procedure
Known amounts of the test sample + labelled
antigen into the microtitre wells
Decant & wash contents of
the well
Measure radioactivity remaining in the
Microtitre wells
[GM counter , Scintillation counter etc]
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incubate
Intensity of radioactivity α 1/ conc. of Ag
in test sample

Advantages & Disadvantages ofAdvantages & Disadvantages of
RIARIA
Advantages
◦ Highly specific: Immune reactions are
specific
◦ High sensitivity : Immune reactions are
sensitive
◦ Possible to detect picograms of Ag
◦ Sepharose beads used in RIA are
reuseable
25

Disadvantages
Radiation hazards: Uses radio labelled reagents
Requires specially trained persons
Labs require special license to handle radioactive
material
Requires special arrangements for
Requisition
storage of radioactive material
radioactive waste disposal.
26

•Both 125
I or 131
I 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).
27

RIA EIA
Sensitivity Nanomolar to
picomolar
Millimolar
Cost More Less
Time duration More Less
Ease of
handling
Tedious Easy
Radiation
hazards
More No
Disposal Care has to be
taken
Easy
Equipment Complex Less
Mechanism Based on the
measurement of
Based on
measurement of28

Applications ofApplications of
RIARIA
 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
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• 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;
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 In Endocrinology
Insulin, HCG, Vasopressin
Detects Endocrine Disorders
Physiology of Endocrine Function
 In Pharmacology
Morphine
Detect Drug Abuse or Drug Poisoning
Study Drug Kinetics
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 Epidemiology
Hepatitis B
 Clinical Immunology
Antibodies for Inhalant Allergens
Allergy Diagnosis
 Oncology
Carcinoembryonic Antigen
Early Cancer Detection and Diagnosis
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 Narcotic drug detection
 tracking of leukemia virus
 diagnosis and treatment of peptic ulcers
 research with Neurotransmitters
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References
•Yalow R, Berson S. Immunoassay of endogenous plasma
insulin in man. J. Clin. Invest 1960; 39: 1157-1175.
•Abraham G. Radioimmunoassay of steroids in biological fluids.
J. Steroid Biochemistry 1975; 6: 261-270.
• Associate Professor Dr. Özhan Eyigör, Uludag University
College of Medicine,Department of Histology & Embryology
Radioimmunoassay
•Sarah Dekat ,NCSS 2006; Radioimmunoassay (RIA):
A Remarkably Sensitive Bioassay
34

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