Radioimmunoassay (RIA) is a sensitive in vitro technique developed in 1959 for measuring antigen concentrations using radioisotopes tagged to antibodies, revolutionizing fields such as endocrinology and blood banking. The method relies on competitive binding between labeled and unlabeled antigens for antibodies, allowing quantification based on radioactivity measurement. RIA is extensively used in clinical practice for hormone level assays and detecting various drugs and diseases.

1. Prepared By: Vipin Kr Shukla
Assistant Lecturer.

2. Radio-Immunoassay:
 Radioisotope is defined as a technique In which
Radio-Isotope is used as a tag or label, (i.e.)
Radioisotope is covalently linked to Antigen or
Antibody for the detection of (AG & AB)
complex.
 Radioimmunoassay (RIA) is a very sensitive in
vitro assay technique used to measure
concentrations of antigens (for example, hormone
levels in the blood) by use of antibodies.

3. Continued…..
 RIA technique is
extremely sensitive and
extremely specific,
requiring specialized
equipment, it remains
the least expensive
method to perform such
tests.

4. History of RIA:
 This technique was developed in the year (1959)
by Endocrinologist (Rosalyn Yalow and Solomon
Berson), to measure the concentration of an Ag &
Ab in the sample.
 In the year (1977), Rosalyn Yalow was awarded
Nobel prize for medicine for the development of
RIA.
 But Rosalyn Yalow could not shared the Nobel
prize because of her certain death.

5. Continued……
 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 in vitro assay .
 The technique of radioimmunoassay has revolutionized
research and clinical practice in many areas, e.g.,
 Blood banking
 Diagnosis of allergies
 Endocrinology

6. Continued……
 In RIA, Radioisotopes are covalently attached to
Ag & Ab.
 We know that Radioisotopes emit the radiations,
which can be measured by using instruments such
as Gamma Counter.
 Most popular Radioisotopes – I- 125.

7. Principle of RIA:

8. Principle of RIA:
 The technique is based on the ability of an
unlabelled form of the substance to inhibit
competitively the binding of a radioactively
labelled substance by specific antibodies .

9. PRINCIPLE OF RADIOIMMUNO ASSAY:
 It involves combination of three principles.
 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)

10. IMMUNE REACTION:
 When a foreign biological substance enters into body blood
stream through non oral route, body recognizes the specific
chemistry on surface of foreign substance as antigen and
produces specific antibodies against the antigen so as nullify
the effects and keep the body safe.
 The antibodies are produced by body immune system so, it is
an immune reaction.
 Here the antibodies or antigens bind move due to chemical
influence.
 This is different to principle of electrophoresis where proteins
are separated due to charge.

11. COMPETITIVE BINDING OR COMPETITIVE
DISPLACEMENT REACTION:
 This is a phenomenon wherein when there are two
antigens which can bind to same antibody, the antigen
with more concentration binds extensively with the
limited antibody displacing other.
 Radiolabelled antigen is allowed to bind to high
affinity antibody.
 Then when patient serum is added unlabelled antigens
in it start binding to the antibody displacing the
labelled antigen.

12. MEASUREMENT OF RADIO EMISSION:
 Once the incubation is over, then washings are
done to remove any unbound antigens.
 Then radio emission of the antigen antibody
complex is taken, the gamma rays from radio
labelled antigen are measured.
 The target antigen is labelled radioactively and
bound to its specific antibodies (a limited and
known amount of the specific antibody has to be
added).

13. Continued……
 A sample, for e.g. blood-serum, is added in order to initiate
a competitive reaction of the labelled antigens from the
preparation, and the unlabelled antigens from the serum-
sample, with the specific antibodies.
 The competition for the antibodies will release a certain
amount of labelled antigen. This amount is proportional to
the ratio of labelled to unlabelled antigen.
 A binding curve can then be generated which allows the
amount of antigen in the patient’s serum to be derived.

14. Continued……
 That means as the concentration of unlabelled
antigen is increased, more of it binds to the
antibody, displacing the labelled variant.
 The bound antigens are then separated from the
unbound ones, and the radioactivity of the free
antigens remaining in the supernatant is measured.

15. Continued…….
 Antigen–antibody complexes are precipitated either by
cross-linking with a second antibody or by means of the
addition of reagents that promote the precipitation of
antigen–antibody complexes.
 Counting radioactivity in the precipitates allows the
determination of the amount of Radiolabelled antigen
precipitated with the antibody.

16. Continued…….
 A standard curve is constructed by plotting the
percentage of antibody-bound Radiolabelled antigen
against known concentrations of a standardized
unlabelled antigen, and the concentrations of antigen in
patient samples are extrapolated from that curve.
 The extremely high sensitivity of RIA is its major
advantage.

18. Process of RIA:
 In RIA, The Antigen & Antibody are labelled with
Radioisotopes.
 We know that the radioisotopes emit the radiations,
these radiations can be detected and measured by
specialized instruments such as Gamma Counter.
 Most popular Radioisotope used as a tag is 125-I (
iodine).
 RIA, determines the concentration of an Antigen,
in a sample based on Competitive binding between
Radiolabelled ( Ag*) & unlabelled (Ag) for its
specific high affinity Antibody.

19. Continued…….
 We add specific Radiolabelled Antigen, to this
well, the amount of these Antigen, are such that
they saturate all the Antigen binding sites, present
in the well.
 Then this well rinsed to remove the unbound
Radiolabelled Antigen.
 At this point if we measure, the Radioactivity of
the well it will be maximum (100%).
 Now, In a second well again we took the same
amount of immobilized Antibody, but this time we
add unlabelled Antigen.

20. Continued…….
 The Antibody, present in the well has same affinity for both labelled
and unlabelled Ag, Since both the (Ag) same, therefore these
antigens will complete with each other for the Antigen binding sites.
 Now, some of the antigens binding sites will be occupied by the
unlabelled Antigens.
 Again after binding takes place the well is rinsed to remove any
unbound antigens.
 Now, measure the radioactivity of the well, it will not be (100%).
 It will decrease because the unlabelled (Ag) have replaced some of
the Radiolabelled antigens and decreasing of the radioactivity tell us
that the (Ag) is present in the sample.

21. RIA as a major clinical tool:
 It is 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.
 Anti-dna antibodies in systemic lupus
erythematosus (SLE).

22. Continued….
 Narcotics (drug) detection
 Blood bank screening for the hepatitis (a highly
contagious condition) virus Early cancer detection.
 Measurement of growth hormone levels tracking
of the leukemia virus.
 Diagnosis and treatment of peptic ulcers research
with brain chemicals called neurotransmitters.