RADIOIMMUNOASSAY (RIA)

DR. RESHMA RAGHU

Department of Biochemistry, SJMCH

RR 1
CONTENTS

Radioimmunoassay

History

Principle of RIA

Technique

Clinical applications

Advantages & Disadvantages

Immunoradiometry (IRA)

RR 2
 IMMUNOASSAY

An assay based on the reaction of an antigen with a specific antibody

RR 3
Immunoassays
All types of immunoassays
revolve around one basic
Antibody
reaction

Antigen

Antibody
RR 4
 Agglutination Works
Antibodies bind to the antigen-coated latex particles.

Antigen

Antibody

RR 5
 Current Technologies

RADIO IMMUNOASSAYS

ENZYME IMMUNOASSAYS

FLUORESCENT IMMUNOASSAYS
CHEMILUMINESCENT IMMUNOASSAYS
RR 6
Development of Immunoassays

RR 7
 Labels in Immunoassays
• Immunoassays require the use of labeled materials in order to measure the
amount of antigen or antibody present.
• A label is a molecule that will react as part of the assay, and in doing so
produce a signal that can be measured in the solution.
• Examples of a label include a radioactive compound, or an enzyme that
causes a change of color in a solution or its fluorescence.

RR 8
 Labels in Immunoassays

• What is the function of the label?

– To provide a means by which the free antigens, or antigen/antibody
complexes can be detected
• What are desirable properties of labels?
– Easily attached to antigen/antibody
– Easily measured, with high sensitivity
– Does not interfere with antibody/antigen reaction
– Inexpensive/economical/non-toxic

RR 9
 Different Labels for Immunoassays

Fluorescein Luminol
125I HRP

RIA ELISA FIA CLIA

RR 10
RR 11
RADIOIMMUNOASSAY (RIA)

In-vitro assay used to

measure the concentration
of antigens using antibodies

Sensitive and extremely

specific test, revolutionised
medicine

Can detect very low

concentrations of ligands in
blood (picograms)

RR 12
 RADIOIMMUNOASSAY (RIA)

Radioimmunoassay (RIA) involves the separation of a protein (from

a mixture) using the specificity of antibody - antigen binding and
quantitation using radioactivity

RR 13
 HISTORY OF RIA

1950s – Rosalyn Yalow & Solomon Berson

Yalow developed technique of RIA for

detecting levels of hormone Insulin in plasma
(1977-NP)

Ultra micro RIA for human TSH – Dr.

Acebedo & Dr. Hayek

RR 14
PRINCIPLE

RR 15
RR 16
RR 17
COMPETITIVE IMMUNOASSAY

RR 18
 Principle of Radioimmunoassay
• Based on immune reaction (antigen-antibody)

• The binding of a radiolabelled Ag* to specific Ab can be partially inhibited

by adding unlabelled Ag

• The extent of this inhibition is a measure of the unlabelled material added.

RR 19
 REQUIREMENTS

• Preparation and characterisation of the antigen

• Radiolabeling of the antigen
• Preparation of specific antibody
• Development of an assay system

RR 20
 Preparation of Antigen

• Isolation from natural sources

• Synthesis of the molecule

RR 21
 Radiolabelling the Antigen
• Most commonly used – Iodine 125
• Other agents – H3, C14, I-131

• Tagging should be done at tyrosine or histidine residues of the protein

molecule

• Tagging should never affect the antigenic properties or antigenic specificity

RR 22
 Preparation of specific Antibodies

• Antigens injected intra-dermally to guinea pigs, specific

antibodies are produced

• Peak antibody response occurs 7-14 days

• Later recovered from their serum samples

RR 23
RR 24
 DEXTRAN- COATED ACTIVATED CHARCOAL METHOD

Antigen-Antibody complex

Dextran coated Dextran coated

activated activated
charcoal charcoal

CENTRIFUGE

ANTIGENS (Ag*)

RR 25
Supernatant with Ag* - Ab complexes
GAMMA COUNTER

RR 26
 Standard Reference Curve for RIA

• To measure the concentration of Ag in a sample, a

reference curve of Ag must be made.

• Ag* + Ab  Ag*-Ab complex + Ag  Ag*-Ab + Ag-Ab +

Ag*
• Ag*-Ab complex separated from Ag*, its radioactivity
measured
• The amount so obtained is plotted against the
concentration of Ag.

RR 27
STANDARD CURVE

RR 28
 WORKING WITH RIA
ADVANTAGES DISADVANTAGES
• Limited assay range
• Highly specific and sensitive • Radio hazards
• Small concentrations can be • Needs high precision
detected • Prolonged reaction time as a
consequence of having to use at
high dilution
• It cannot distinguish between the
active protein molecules and
biologically inactive proteins which
may be antigenic.
• Difficulty to automate
• Lack of direct linear relationship
between the analyte and signal
response

RR 29
 DIAGNOSTIC USES OF RIA

• For measuring/analysis of hormones, vitamins and diagnostic markers –

Insulin, TSH, vitamin B12, CEA

• Therapeutic drug monitoring – Digoxin, Morphine, Barbiturates

• Diagnostic in detecting infections – Hepatitis B antigen

• Certain antibodies like anti DNA antibody in SLE

RR 30
RR 31
 IMMUNORADIOMETRY (IRA)

• Variant of RIA

• Done in cases where Radiolabelled Ag is not available

• Label antibody instead of antigen

• Radioactivity is measured

RR 32
RR 33
References:

1. Clinical chemistry – TEITZ

2. Clinical Chemistry – KAPLAN
3. Biophysics – UPADHYAYA
4. Molecular techniques - Wilson & Walker

RR 34