Radioimmunoassay (RIA) is an in vitro technique used to measure hormone concentrations in blood using antibodies. It involves radiolabeling a known quantity of the antigen/hormone and using its competition with unlabeled antigen for a limited number of antibody binding sites. This allows measurement of unlabeled antigen concentrations in unknown samples by comparison to a standard curve. RIA offers high sensitivity to detect picogram quantities but requires radioactive materials that require special handling and disposal. Quality control measures precision, sensitivity and specificity.

1. ESTIMATION OF HORMONES BY RIA
(Radioimmunoassay)
Submitted to Submitted by
Dr. M. Narayana Swamy V.Praveen
Professor & Head MVHK 1648
Dept. of Veterinary Physiology
Veterinary College of Bangalore
Hebbal

2. Introduction
• 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
• For this method one need a pure preparation of known
antigen / antibody, or both, in order to standardize the assay.
• 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.

3. Principle
• The principle of RIA is based on the theory that in the absence
of an unlabeled antigen or hormones (H), the labeled
radioactive hormones (H*) has maximal opportunity to react
with limited number of antibody binding site(Ab).
• Antibody have bivalent binding site so one antibody molecule
can bind two hormone molecules.
• If some of the limited antibody combining sites are allowed
to react with the unlabeled hormones, fewer sites will be left
to react with the labeled hormones, which results in a
decrease in antibody – bound radioactivity.

4. Contd…
• This data may be graphed on an arithmic scale and a standard
curve is made.
• The amount of hormone in unknown sample is determined by
comparisions with the standard curve.
Ag + Ag* + Ab  AgAb + Ag*Ab + Ag + Ab*
[Ag : ligand to be measured ; Ag* radiolabelled ligand]
Uses an Antigen – Antibody reaction to estimate a
ligand

5. Requirements
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

6. Preparation & Radiolabelling of the
Antigen
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
◦ Tagging should NOT affect Antigenic specificity &
Antigenic activity !

7. Preparation of the Specific Antibody
• Antigen injected intradermally into rabbits or guinea
pigs  antibody production
• Antibodies recovered from the serum
• Some ligands are not Antigenic
– Hormones, Steroids, Drugs  HAPTENS
– Eg: Gastrin, Morphine,
– Haptens conjugated to albumin  antigenic

8. Development of the Assay System
• A crucial step is separation of unbound antigens
• This achieved by binding the antibodies to the 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

9. Assay Procedure
• Add known amounts of the test sample + labelled antigen
into the microtitre wells
• Incubate  allow the reaction to reach completion
• Decant & wash contents of the well  removes all unbound
antigens
• Radioactivity remaining in the Microtitre wells measured by a
Counter [GM counter , Scintillation counter etc]
• Intensity of radioactivity is inversely correlated with the conc
of antigens in the test sample
• Sensitive to very low conc. of antigens

10. Advantages
• Radioimmunoassay is widely-used because of its great
sensitivity.
• Using antibodies of high affinity, it is possible to detect a few
picograms (10−12 g) of hormone in the tube.
• The greater the specificity of the antiserum, the greater the
specificity of the assay

11. Limitations
• The main drawbacks to radioimmunoassay are the expense
and hazards of preparing and handling the radioactive
antigen.
• Both 125I or 131I 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).

12. RIA
 Advantages
◦ Flexibility
◦ Sensitivity
◦ Size
 Disadvantages
◦ Toxicity
◦ Shelf life
◦ Disposal costs

13. SCHEMATIC REPRESENTATION OF RIA

14. STANDARD CURVE FOR RIA

15. QUALITY CONTROL PARAMETERS IN
RIA
• In the process of hormone estimation the quality control in
terms of sensitivity, specificity and precision are required to
be carried out for each hormone.
• Sensitivity – it is generally regarded as hallmark of good assay.
Its defined as minimum amount of hormone (lowest
detectable limit) distinguishable from zero concentration.
• Precision – Its also known as reproducibility, is a measure of
variation observed between repeated determination of the
same sample in different assay.
• Specificity – Specificity of an assay is defined as the degree to
which an assay responds to substance other than for which
the assay is designed.

16. INSTRUMENTATION

17. Thank you