2. CONTENTS
• HISTORY
• INTRODUCTION
• PRINCIPLE
• INSTRUMENTATION
• REQUIREMENTS OF RIA
• METHODOLOGY OF THE ASSAY
• PROCEDURE
• ADVANTAGES
• DISADVANTAGES
• APPLICATIONS
3. HISTORY
Radioimmunoassay (RIA) is a very sensitive in vitro
assay technique used to measure concentrations of
antigens by use of antibodies
RIA was primarily developed by Solomon Berson &
Rosalyn Yalow(1959) for the quantitative
measurement of insulin in human plasma.
4. In 1977 Yalow received the Nobel Prize in Physiology or Medicine for her
and Berson’s development of RIA as it applied to tracing hormones in the
body.
5. Rosalyn Yalow in
the laboratory.
She conducted
her research at
the VA Medical
Center in Bronx,
New York, from
1947 to 1991.
6. Since Nobel Prizes are only given to the living, Yalow received the award without
Berson, who died in 1972. She shared it with two other scientists, who were honored
for their work on hormone production in the brain.
Rosalyn
Yalow
receives the
Nobel Prize
in
Physiology
or Medicine
1977 from
King Carl
XVI Gustaf
of Sweden.
7. INTRODUCTION
“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.
9. “RADIOIMMUNOASSAY IS A TECHNIQUE FOR
DETERMINING ANTIBODY LEVELS BY
INTRODUCING AN ANTIGEN LABELED WITH A
RADIOISOTOPE AND MEASURING THE
SUBSEQUENT RADIOACTIVITY OF THE
ANTIBODY COMPONENT”.
10. RIA is specific, sensitive, & rapid assay
technique.
The major disadvantage of RIA is health &
safety risks by the use of radiation &
maintaining licensed radiation & disposal
program is difficult.
11. The technique of radioimmunoassay has
revolutionized research and clinical practice in many
areas, e.g.,
• Drug detection from blood samples
• Blood bank screening for contaminants,e.g. The hepatitis virus
• Detection of cancer biomarkers
• Measurement of hormone levels
• Measurement of disease associated antigen levels
• Measurement of vitamins or other proteinaceous antigen level.
• Diagnosis of allergies
12. OBJECTIVE
The objective is to determine the
concentration of a unlabelled antigen.
So in order to conduct RIA a standard
curve is made with concentration versus
% of radioactive compound bound.
14. PRINCIPLE
Radioimmunoassay (RIA) involves the separation
of a protein (from a mixture) using the specificity
of antibody - antigen binding and quantitation
using radioactivity.
15. PRINCIPLE
THE TECHNIQUE
The Principle of Radioimmunoassay (RIA)
An immune reaction i.e. antigen, antibody binding.
• A competitive binding or competitive displacement
reaction. (It gives specificity)
• Measurement of radio emission. (It gives sensitivity)
16. 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.
17. serum
A Antigen
1. Anti A
Antibodies
2. Radiolabeled
A antigen
3. Unlabeled
A antigen
Radioisotopes
125I Tritium
18. 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.
The radioactivity of each is measured.
20. INSTRUMENTATION
• MICRO TITER PLATE :- A micro titer plate is used
mostly used for this assay. A microtiter plate could
have 6,24,96,384 or even sometimes 1536 wells
arranged in rows.Each well of a microplate can only
hold very small amount of liquid.
• PURE ANTIGEN :- Antigen may be obtained from
biological sample or by synthetic form it should be
pure. It is used as standard or calibrator.
• RADIO LABELLING OF ANTIGEN :- The most
commonly used radiolabels in RIA are tritum and
iodine.They have adequate activity and have long
enough half lifes.
21. INSTRUMENTATION
• ANTIBODY :- specific antibodies are obtained by
injecting the Ag to animals.
• STANDARDS :-
% antibody bound with
antigen in unknown
sample
Amount of antigen in
unknown sample
22. INSTRUMENTATION
• CENTRIFUGE :- Used for the separation of
precipitated and supernatant liquid from the sample.
Centrifuge
Swing Bucket
Rotor
Pellet is formed
at the bottom of
the test tube
Range
1200-2500 rpm
Fixed Angle
Head Rotor
pellet is
formed at an
angle
Range
3500-4000 rpm
24. INSTRUMENTATION
• RADIO ACTIVE COUNTERS :-
Two types of counters are used namely,
1.Gamma counter
2. Scintillation counters
• Gamma counter:- These are used for the gamma
energy emitting isotopes
E.g. 125I .
• Scintillation counter :- These are used for counting
beta energy emitting isotopes
E.g. Tritium 3H & 14C Isotopes
25. REQUIREMENTS OF 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
26. REQUIREMENTS OF RIA
1. PREPARATION & RADIOLABELLING OF THE AG
Antigens prepared by,
• Synthesis of the molecule
• Isolation from natural sources
27. 2. RADIOLABELLING [TAGGING PROCEDURE]
3 H , 14 C, 125 I are used as radioactive tags
Antigens are tagged to 3 H , 14 C, 125 I
NOTE: Tagging should NOT affect Antigenic
specificity & Antigenic activity !
28. REQUIREMENTS OF RIA
3. PREPARATION OF THE SPECIFIC ANTIBODY
Antigen injected intradermally into rabbits or
guinea pigs to induce antibody production
Antibodies recovered from the serum
29. REQUIREMENTS OF RIA
4. DEVELOPMENT OF THE ASSAY SYSTEM
Crucial step is separation of unbound antigens
Ab 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 Ag
30. METHODOLOGY OF THE ASSAY
In RIA the sequential steps followed are:
Mix a fixed concentration of antiserum containing
specific antibody with a constant quantity of
radiolabelled antigen.
Incubate it for specified duration & at an appropriate
temperature usually +4°c.
A definite volume of sample containing unlabelled
antigen to be measured is added to the reaction test
tube.
31. METHODOLOGY OF THE ASSAY
The antibody reacts with both radioactive & unlabelled
antigen forming an ab-radiolabelled antigen
complexes.
Both radioactive & unlabelled antigens are more or
less same immunochemically, they will compete for
limited number of antibody sites available.
The radio activity falls because the unlabelled antigen
dilutes i.e.)reducing the no of labelled antigen
combining with antibody.
The counts obtained from the radioactivity are used to
determine the unlabelled antigen concentration in the
sample, the interpretation being done on the standard
curve.
32. PROCEDURE
1. A specific quantity of antigen is made radioactive.
2. The radiolabeled antigen is mixed with a specified
amount of antibody for a particular antigen causing
the two to bind to each other.
3. A serum sample from a patient that contains an
unknown quantity of the same antigen is added
causing the unlabeled antigen from the serum to
compete with the radiolabeled antigen for the
antibody binding sites.
33. PROCEDURE
4. The unlabeled antigen concentration increases
causing it to bind more to the antibody.
5. The radiolabeled variant is displaced reducing the
ratio of antibody-bound radiolabeled antigen.
6. The bound antigen separates from the unbound
ones.
7. The remaining free antigen’s radioactivity in the
supernatant is measured.
34. PROCEDURE
Microtiter plate is taken
Coated with anti A monoclonal
antibodies
Radiolabeled antigens are added
to the well in excess quantity
No antibody is left free ; some
radiolabeled antigens are left
unbound
35. Unbound antigens are removed
with the help of washing
Now Radioactivity in the antibody
of the well is 100%
A very small but known amount of
unlabeled antigen is added
This addition will create
competition between radio
labeled & unlabeled antigen
Few radio labeled antigens are
removed from the antibodies
Addition of unlabeled antigen
Radiolabeled Ags
decreased
Due to the addition
of unlabeled Ag
36. 0 ng 100%
1 ng 90%
2 ng 80%
3 ng 70%
4 ng 60%
5 ng 50%
6 ng 40%
7 ng 30%
8 ng 20%
9 ng 10%
10 ng 0%
38. 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 reusable
39. DISADVANTAGES
1. Radiation hazards: Uses radio labelled reagents
2. Requires specially trained persons
3. labs require special license to handle radioactive
materials
4. Requires special arrangements for,
• Requisition, handling, Storage of radioactive material
• Radioactive waste disposal.
5. Expensive instrumentation for the conting of
radioactivity, Both 125I or 131I emmit gamma radiation
that requires
special counting equipment.
40. APPLICATIONS
1. Analysis of hormones, vitamins, metabolites,
diagnostic markers
Examples:-
• ACTH, FSH, T3, T4, Glucagon, Insulin,Testosterone
• vitamin B12, prostaglandins, glucocorticoids,
2. Therapeutic drug monitoring:
Examples:- Barbiturates, morphine, digoxin,
3. Diagnostic procedures for detecting infection
Examples:- HIV, Hepatitis A, B etc
41. 4. Used to assay
Plasma levels of:
• Most of our hormones;
• Digitoxin or digoxin in patients receiving these drugs;
• Certain abused drugs
Screening donated blood
• Hepatitis B
• Hepapitis C
• HIV
42. 5. In Endocrinology
• Insulin, HCG, Vasopressin
• Detects Endocrine Disorders
• Physiology of Endocrine Function
6. In Pharmacology
• Morphine
• Detect Drug Abuse or Drug Poisoning
• Study Drug Kinetics
43. 7. Clinical Immunology
• Antibodies for Inhalant Allergens
• Allergy Diagnosis
8. Early Cancer Detection and Diagnosis
9. Narcotic drug detection
10. Tracking of leukemia virus
11. Diagnosis and treatment of peptic ulcers
12. Measuring “rheumatoid factors” and other auto
antibodies in autoimmune disease likeluous
erythematosus.
44. RECENT APPLICATIONS
• Refinement of zalcitabine pharmacokinetics
• Estradiol measurement in translational studies of
breast cancer.
• Significance of prostate specific antigen in prostate
cancer patients & in non cancerous prostetic disease
patients.
45. REFERENCES
Yallow R, Berson . Immunoassay Of Endogeneous Plasma Inslin In
Man. J.Clin. Iinvest 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
