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).
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.
