Radio Immuno Asssay

1. Mangesh G Jadhav
Roll no- 08
Fergusson college ,pune
RadioImmuno Assay

2. Immuno-Assay
 Immuno- it refers to the response that cause
body to generate antibodies.
 Assay- refers to test.
 Immunoassay is a test that use immuno-
complexing when antigen and antibodies are
brought together.

3. CATEGORIES of
IMMUNOASSAY
 Competitive
 Non-competitive
 Homogenous
 Heterogenous

4. Competitive assay
 Unlabelled antigen in the test sample is
measured by its ability to compete with
labelled antigen in the immuno-assay.
 In this assay, less labelled antigen measured
means more of the (test sample) unlabelled
antigen is present.

6. Non-Competitive Assay
 This type gives the highest level of sensitivity and
specificity.
 These are normally used to measure critical
analytes such as hepatitis and cardiac markers.
 The measurement of the labelled anti-bodies is
directly proportional to the amount of antigen

8. Homogeneous And Heterogenous
Assay
 The assay which requires separation of bound
Ab-Ag* complexes are referred as
Heterogenous immuno-assay.
 The assay which do not require separations
are Homogenous immuno-assay.
 Homogenous assays are generally applied to
the measurement of small analytes such as
Ab used as therapeutic drugs.

9. Types of Immuno-Assay
 Radio-Immuno Assay (RIA)
 Enzyme-Immuno Assay (ELISA)
 Fluroscence Polarization Immuno Assay
(FPIA)

10. History
 RIA was introduced by two
endocrinologists S. A.
Berson and Roslyn Yalow
(1960) determining level of
insulin- anti insulin
complexes in diabetics.
 It represented first method
to detect in-vitro hormone
level in blood.
Rosalyn Yalow became
first lady to win a Nobel
Prize with her work on
RadioImmuno Assay.

11. Principle
 Radio-Immuno Assay (RIA) involves competitive
binding of radio-labeled antigen and unlabeled
antigen to a high-affinity antibody.
 it involves separation of protein (from mixture)
using specificity of Ag-Ab and quantification using
radioactivity.

12.  The antigen is generally
labelled with a gamma-
emitting isotope such as
125I, but beta-emitting
isotopes such as tritium (3
H) are also routinely used
as labels.
 This radiation is measured
by beta or gamma counters.

13. Technique
 The mixture is prepared of:
 Radioactive antigen
 Antibodies against that antigen
 Known amount of unlabeled (cold) antigen is
added to the samples of the mixture. These
competes for binding to the sites of the
antibodies.

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

16. Gamma Counter

17. Requirements of RIA
 Preparation and characterization of antigen
(ligand to be analysed).
 Radiolabelling of antigen.
 Preparation of specific antibody.
 Development of assay system.

18. Preparation & Radiolabeling Of
Ag
 Antigen preparation by:
 Molecule synthesis
 Natural source
 Radiolabeling (Tagging procedure)
 3H, 14C, 123I are used as radio active tags
 Anitgens are tagged to 3H, 14C, 123I .
(Tagging should not affect the specificity and
activity of the Antigen).

19. Preparation of specific
Antibody
 Antigen injected intra dermally to rabbits or
guinea pigs  antibody production.
 Antibodies recovered from the serum.

20. Development of Assay System
 Crucial step is separation of unbound
antigens.
 Antibodies bind to the micro-titer well surface
(Solid surface RIA).
 Antigens bound to the fixed antibodies remain
stuck to the inner surface.
 Decanting & washing the wells remove
unbound antigens.
 Other techniques of separations:
centrifugation, precipitation and
electrophoresis.

21. Assay Procedure
 Known amount of the test sample + labelled
antigen in micro-titer well plate
incubation
 Decant and wash contents of the well
 Measure the radioactivity remaining in the wells
(GM counter, Scintillation counter, etc)
Intensity of radioactivity is inversely proportional to
the concentration of antigen in test sample.

22. Advantages of RIA
 Highly specific: Immune reactions are highly
specific.
 High sensitivity: Immune reactions are sensitive.
 Possible to detect “picograms” of antigen.
 Sepharose beads used in RIA are reusable.

23. Disadvantages
 Radiation hazards: uses radio-labelled reagents.
 Requires specially trained person.
 Lab requires special license to handle radioactive
material.
 Require special arrangements for:
 Requisition.
 Storage of radioactive material.
 Radioactive waste disposals.
 Both 131I and 125I emit gamma radiations which that
requires special counting equipments
 The body concentrates iodine atoms (radioactive or
not) in the thyroid gland where they are incorporated
into thyroxine (T4).

24. Applications of RIA
 Analysis of hormones, vitamins, metabolites,
diagnostics markers
 Eg. ACTH, FSH, T3, T4, Glucagon, Insulin,
Testosterone, vitamin B12, prostaglandins,
glucocorticoids.
 Therapeutic drug monitoring:
 Barbiturates, morphine, digoxin.
 Diagnostic procedure for detecting infection:
 HIV, Hepatitis A, B, etc.

25.  RIA has become a major tool in clinical
laboratories where it is used to assay:
 Plasma levels of-
 Most of hormones,
 Digitoxin and 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).

26.  In Endocrinology:
 Insulin, HCG, Vasopressin
 Detect Endocrine Disorders
 Physiology of Endocrine Function
 In Pharmacology:
 Morphine
 Detect drug abuse or drug poisoning
 Study of drug kinetics

27.  Epidemiology:
 Hepatitis B
 Clinical Immunology:
 Antibodies for Inhalant Allergens
 Allergy diagnosis
 Oncology:
 Carcinoembryonic Antigen
 Early cancer detection and diagnosis

28.  Narcotic drug detection.
 Tracking of leukemia virus.
 Diagnosis and treatment of peptic ulcers.
 Research of neurotransmitters.
 Detecting allergens present in food and house
dust
 RAST (Radio AllergoSorbent Test) to detect specific
IgE antibodies to suspected or known allergens. IgE
antibody associated with type I allergic response:
Pollen ( fine to coarse powder containing
microgamatophytes of seeds).
 The amount of radioactivity is directly proportional
to serum IgE for the allergen.