 Introduction

 Principle of RIA
 Theory
 Requirements
 Methods
 Merits & De-Merits
 Applications
 Related Techniques
 References
 Radioimmunoassay (RIA) is a sensitive
method for measuring very small
amounts of a substance in the blood.
 Radioactive versions of a substance, or
isotopes of the substance, are mixed
with antibodies and inserted in a
sample of the patient's blood.
 The same non-radioactive substance in
the blood takes the place of the isotope
in the antibodies, thus leaving the
radioactive substance free.
 Theamount of free isotope is then
measured to see how much of the original
substance was in the blood.

 Thisisotopic measuring method was

developed in 1959 by two Americans,
biophysicist Rosalyn Yalow (1921-) and
physician Solomon A. Berson (1918-1972).
 Yalow and Berson - first radioisotopic
technique - to study blood volume and
iodine metabolism. They later adapted
the method to study how the body uses
hormones, particularly insulin, which
regulates sugar levels in the blood.
 The researchers proved that Type II
(adult onset) diabetes is caused by the
inefficient use of insulin. Previously, it
was thought that diabetes was caused
only by a lack of insulin.
 • In the year 1959, Drs.
Rosalyn Yalow & Soloman
Berson invented the
radioimmunoassay, which
Dr. Rosalyn Yalow became the first
applied the use of female to win a Nobel Prize with
radioisotopes in the her work on the radioimmunoassay.

measurement of
insulin.

• The RIA is the

predecessor of modern
immunoassays.
 Radioimmunoassay (RIA) analysis -
RIA involves the separation of the
drug using the specificity of antibody
- antigen binding and quantitation
using radioactivity
 Radioimmunoassay is based on the
antigen-antibody reaction in which tracer
amounts of the radio-labeled antigen
competes with endogenous antigen for
limited binding sites of the specific
antibody against the same antigen.

 In principle, radio-labeled antigen

should be similar in bio-activity and/or
immunoreactivity of the native antigen
Ag + Ag* + Ab  AgAb + Ag*Ab + Ag* +
Ag

› Unbound Ag* and Ag washed out

› Radioactivity of bound residue measured
› Ligand conc is inversely related to
radioactivity

[Ag : ligand to be measured ; Ag*

radiolabelled ligand]
A fixed concentration of radio-labeled
antigen in trace amounts - incubated
with a constant amount of antiserum

-total antigen binding sites on the

antibody are limited such that the only
30–50% of the total radio-labeled
antigen may be bound in the absence
of the antigen -
 When unlabeled antigen, either as
standard or test sample, is added to
this system, there is competition
between radio-labeled antigen and
unlabeled antigen for the limited
constant number of binding sites on
the antibody.

 The amount of radio-labeled antigen

bound to antibody decreases as the
concentration of unlabeled antigen
increases.
 The radioactivity in the labelled
antigen-antibody complex is measured
after separating the bound complex
from the free antigens by suitable
separation technique.

 The counts obtained are used to

determine the unknown antigen conc.,
by interpreting on the standard curve.
 The method of assaying the
radioactivity of the bound and/or
unbound fraction solely depends on the
nature of isotope & separation method.
 The expt. Cond. such as – pH, ionic
comp.- protein content or any other
interfering factors should be identical
for std and sample.
A highly purified labelled antigen
 A specific antiserum
 A method for separation
 A method for Quantitation
 Radiolabelling [Tagging procedure]

› 3H 14
C 125
I are used as radioactive
tags
› Antigens are tagged to 3 H 14 C 125
I
› Tagging should NOT affect
Antigenic specificity &
Antigenic activity!!!
 Usually high specific activity
- radio-labeled (125-I) antigen is used -
prepared by
- iodination of the pure antigen on its
tyrosine residue(s)
- by chloramine-T or peroxidase methods
and then separating the radio-labeled
antigen from free-isotope by gel-filtration
or HPLC.
 Some ligands are not Antigenic
› Hormones, Steroids, Drugs  HAPTENS
› Eg: Gastrin, Morphine,

Haptens conjugated to albumin  antigenic

 Antigen injected intradermally into
rabbits or guinea pigs  antibody
production

 Antibodiesrecovered from the serum,

and stored in non-defrosting freezer at
≤ 20°C or in liquid nitrogen at -196°C
and used.
 Followoptimal incubation condition
e.g. buffer, pH, time and temperature,
while separation.

 Various
separation methods are used
based on physical and biochemical
characters of the bound complexes.
 Precipitation of the antibody
- Fractional Precipitation
› Centrifugation
› Filtration
 Adsorption of the free Antigen
 Use of solid phase Antibody
 Gel Filtration
 Adsorption Chromatography
 Partition Chromatography
› Dialysis
 Electrophoresis
 Ultrafiltration
 Precipitation of the antibody:
 Used if mol. Size of B & F forms of Ag
differ considerably

 Chemically – ethanol, dioxan,

isopropanol, PEG, Amm.sulfate etc.

 Double Ab technique – A secondary

antibody is used to precipate.
 Adsorption of free antigen:

 Charcoal coated with dextran is

often used

 Talc,cellulose and ion exchange

resins also used.
 Use of solid phase Ab:

 Antibody is linked to solid phase

– initial reaction – heterogenous
system
- after equilibrium – separation
simple.

 Dextran,acrylamide resins, inorg.

Metal oxides (Si, Al, Ti) – controlled
pore beads form – used.
Then,

 Electrophoresis
 Gel Filtration
 Adsorption Chromatography
 Fractional Precipitation
› Centrifugation
› Filtration
 Partition Chromatography
› Dialysis are used…
 Variousinstruments are used –
detection and measurement of
radioactivity.

› Scintillation counter
› Geiger-muller counter
› Gas counters etc..
 Bothfree(F) and bound(B) fractions are
counted,

 Variousrelationships are established

between the variables and
mathematical equations are defined for
calculating required data and
quantified.
 Competitive Immunoassays

 Non-Competitive Immunoassays
 unlabelledanalyte in the test sample is
measured by its ability to compete with
labeled antigen for a limited number of
antibody binding sites.

-less label measured in the assay

means more of the unlabeled (test
sample) antigen is present.

 The
amount of antigen in the test
sample is inversely related to the
amount of label measured in the
competitive format. As one increases,
 Highest level of assay sensitivity and
specificity.

 This
format is referred to as a
“sandwich” assay because the
analyte is bound (sandwiched)
between two highly specific antibody
reagents.
 The reaction mixture typically includes an
excess of labeled antibody, so that all
drug/metabolite is bound. The amount of
antibody-antigen complex is then
measured to determine the amount of
drug present in the sample. The
measurement of labeled analyte, usually
antibody, is directly proportional to the
amount of antigen present in the sample.
 Immunoassay methods that require
separation of bound Ab-Ag* complex -
heterogeneous immunoassays. Those
that do not require separation -
homogeneous immunoassays.

 Homogeneous - generally applied -

measurement of small analytes -
abused and therapeutic drugs. Since -
do not require the separation - much
easier and faster to perform.
 great sensitivity
 possible
to detect a few picograms
(10−12 g) of antigen.
 greater specificity of the assay.
 Expensive

 hazards in preparing & handling the

radioactive Ag.
 Requires special counting equipment
 Thebody concentrates iodine atoms —
radioactive or not — in the thyroid
gland where they are incorporated in
thyroxine.
 Pharmacy
› Quantification of drugs in serum
› Detect Drug Abuse or Drug Poisoning
› Study Drug Kinetics
Novel uses:
› In vitro hepatic metabolic activity
› Stereospecific quantification – D & L forms.
› Measuring – hormones, mediators, growth
factors, cytokines, prostanoids etc..
 Endocrinology
› Insulin, HCG, Vasopressin
› Detects Endocrine Disorders
› Physiology of Endocrine Function

 Epidemiology
› Hepatitis B
 Clinical Immunology
› Antibodies for Inhalant Allergens
› Allergy Diagnosis

 Oncology
› Carcinoembryonic Antigen
› Early Cancer Detection and Diagnosis
 Enzyme Multiplied Immunoassay (EMIT )

 Enzyme-linked immunosorbent
assay (ELISA)

 Fluorescent Polarized Immunoassay

 thedrug in the sample & the drug
labeled with G6PD compete for
antibody binding sites.

 Binding inhibits enzyme activity,

while free enzyme remains active to
interact with.

 Enzyme activity/absorbance is
directly proportional to drug
 - competitive, heterogeneous EIA

 Reactioncomponents are absorbed or

bound to the surface of a solid phase,
commonly a well of a microtiter plate

 Absorbance is measured using a micro-

plate reader

 Sample absorbance is inversely

proportional to drug concentration
 the drug in the sample competes with
fluorescein-labeled drug for antibody
binding sites.

 Reaction mixture is excited by

planepolarized light.

 As the tracer returns to a lower energy

state, it emits light; polarization is
measured.

 The polarization value of the sample is

 Pharmaceutical analysis – Ashutoshkar
 Biochemistry - Vasudevan
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