Radioimmunoassay (RIA) is a highly sensitive in vitro technique developed to measure antigen concentrations in samples, notably introduced for insulin detection in 1960. The method relies on competitive binding between radiolabeled and unlabeled antigens with specific antibodies, allowing precise quantification of hormones and other substances. While RIA is widely used for its specificity and sensitivity in various clinical applications, it also poses radiation hazards and requires specialized training and equipment.

1. Radioimmuno Assay
Presented by : Azad Rawat

2. Radioimmunoassay
• 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. • RIA technique is extremely sensitive and
extremely specific, requiring specialized
equipment, it remains the least expensive
method to perform such tests

4. • 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.

5. • The technique of radioimmunoassay has
revolutionized research and clinical practice
in many areas, e.G.,
• Blood banking
• Diagnosis of allergies
• Endocrinology

6. Method
• To perform a radioimmunoassay, a known
quantity of an antigen is made radioactive,
frequently by labeling it with gamma-
radioactive isotopes of iodine attached to
tyrosine (hot).
• This radiolabeled antigen is then mixed with
a known amount of antibody for that
antigen, and as a result, the two specifically
bind to one another

7. • Then, a sample of serum from a patient
containing an unknown quantity of that
same antigen is added.
• This causes the unlabeled (or "cold")
antigen from the serum to compete with the
radiolabeled antigen ("hot") for antibody
binding sites.

8. •Principle :
• 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. The principle of RIA
• The amount of Ab per tube is kept
constant, the amount of antigen added
(known or unknown) is the variable
parameter.
• The added antigen will be distributed
between a bound (B) and a free (F)
fraction. This distribution is governed by the
association constant (KA) of the Ab:
Ab + Ag 􀁺 AgAb and K = [AbAg]
/[Ab][Ag]

10. • Conclusion: If total Ab input is kept
constant, the value of B/F is a measure
for the total Ag input

13. Requirements for the development
of an RIA
1. Pure antigen : for - standards (μg),
- Tracer production (tens of μg)
- Ab production (hundreds of μg)

14. 2. Tracer : self-made or commercial.
3. Specific, high-affinity antibody : self-
made or commercial.
4. A method to separate bound and free
antigen.
5. (Optional) : A system to extract the
antigen from the sample.

15. USES
Radioimmunoassay is widely-used because
of its great sensitivity.
The greater the specificity of the antiserum,
the greater the specificity of the assay.

16. Advantages & Disadvantages of RIA
• 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

17. 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.

18. Applications of RIA
 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