RADIO IMMUNO ASSAY

RADIOIMMUNO ASSAY
PRESENTED BY,
SURIYAPRIYA .K,
1st yr M.PHARM
DEPARTMENT OF PHARMACEUTICAL ANALYSIS,
ADVANCED PHARMACEUTICAL ANALYSIS,
KMCH COLLEGE OF PHARMACY,
COIMBATORE.
KMCH COLLEGE OF PHARMACY
1
7/25/2021

OVERVIEW
 IMMUNOASSAY
 ANTIBODY ,ANTIGEN, ANALYTE
 RADIOIMMUNOASSAY INTRODUCTION
 HISTORY
 PRINCIPLE
 REQUIREMENTS FOR ASSAY
 STEPS INVOLVED IN RIA
 PROCEDURE INVOLVED IN RIA
 INSTRUMENTATION
 RIA IN DIGITALIS
 RIA IN INSULIN
 MERITS OF RIA
 DEMERITS OF RIA
 DRAWBACK
 REFERENCE 7/25/2021
KMCH COLLEGE OF PHARMACY 2

IMMUNO ASSAY
 An immunoassay is a biochemical test ,that measures the presence or
concentration of a macromolecule or a small molecule in a solution through
the use of an antibody or an antigen.
 An immunoassay is a test that uses antibody and antigen complexes .
 An antibody and antigen complex is also known as an immune- complex .
 refers to an immune response that causes the body to generate
antibodies .
 refers to a test .
“Immuno”
“Assay”
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3

TYPES OF IMMUNO ASSAY
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ANTIBODIES, ANTIGENS AND ANALYTES
An antibody is a protein that is produced by the body immune response to an
“invading” (foreign) substance.
Antibodies are produced as part of the body’s immune response to protect
itself.
An antigen is the substance that the body is trying to “fight off” by mounting an
immune response.
For example, Drug is the antigen that binds to the antibody.
An immunogen is a substance that elicits immune response.
E.g. drug-protein conjugate.
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 An analyte is anything measured by a laboratory test.
 In immunoassay testing, the analyte may be either an antibody, or an antigen.
Immunoassays utilize one or more selected antibodies to detect analytes of
interest.
The analytes being measured may be:-
1. That are naturally present in the body (such as a thyroid hormone)
2. The body produces but are not typically present (such as a cancer antigen)
3. Do not naturally occur in the body (such as an abused drug)
TO BE CONTINUED ,
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6

STRUCTURE OF
ANTIBODIES
 Antibodies are produced by the B lymphocytes , Y
shaped (Immunoglobulin )
 The most important one is immunoglobulin G
(IgG).
 IgG is a protein composed of two main structural
and functional regions:
Fab region: Contains the antigen (Ag) binding
site that varies between different antibodies.
Fc region: Region of constant structure within an
antibody class.
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RADIOIMMUNO ASSAY
 Radioimmunoassay is a technique for determining antibody levels by
introducing an antigen labelled with a radioisotope and measuring the
subsequent radioactivity of the antibody component.
 For example, Hormone levels in blood (insulin ) by use of antibodies .
 The RIA technique is extremely sensitive and extremely specific .
 It is the technique used for the estimation of any ligand / antigen in
biological fluids ,based on antigen antibody reaction .
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HISTORY
 Developed in 1959 by Rosalyn Yalow and Solomon Berson
for measurement of insulin in plasma.
 Rosalyn Yalow was a nuclear physicist.
 She developed radioimmunoassay (RIA) together with
doctor Solomon Berson
 It represented the first time that hormone levels in the blood
could be detected by an in vitro assay.
 In 1977 Yalow received the Nobel Prize for her and Berson’s
development of RIA
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REQUIREMENTS
 Micro titer plate / Polypropylene Test tubes
 Pure antigen
 Pipette
 Radio labelled antigen
 Antibodies
 Standard’s / Reference
 Vortex mixer
 Centrifuge
 Radioactive counter
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RADIO IMMUNO ASSAY
TWO METHODS EMPLOYED
FOR DRUG DETECTION IN BIOLOGICAL
MATRICES
WITH DOUBLE ANTIBODY RIA WITH COATED TUBE RIA
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PREPARATION AND RADIOLABELING OF THE ANTIGEN
Antigens prepared by
synthesis of the molecule
isolation from natural sources
Radiolabeling [Tagging procedure ]
3H,14C,125I are used as radioactive tags
Antigens are tagged to 3H , 14c , 125I
Tagging should NOT affect antigenic specificity and activity 7/25/2021
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PREAPRATION OF SPECIFIC ANTIBODY
 Antibody can be either polyclonal or monoclonal .
 Antigen injected intradermally into rabbit or guinea pigs antibody
production .
 Antibodies recovered from the serum .
 Some ligands are not antigenic.
 With the low molecular weight substances such as the thyroxine, steroid
hormones, and drugs conjugation is essential ..
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POLYCLONAL ANTIBODY MONOCLONAL ANTIBODY
Cheap to produce Expensive to produce
Mixed population of
antibodies
Single antibody species
May bind to different areas
of the target molecules
Will only bind single
specific site
Tolerant of small changes in
protein structure
May recognise a particular
protein form
Monoclonal antibody
Polyclonal antibody
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PRINCIPLE OF RIA :
 The basic principle of radioimmunoassay is competitive binding, where a radioactive antigen
("tracer") competes with a non-radioactive antigen for a fixed number of antibody or receptor binding
sites.
 The radio labeled antigen should have similar biological activity and immunogenicity like that of
native antigen .
LABELED ANTIGEN + SPECIFIC ANTIBODY LABELED ANTIGEN Ag *ANTIBODY complex
Ag* Ab Ag*Ab
UNLABELED ANTIGEN
Ag
UNLABELED ANTIGEN - ANTIBODY COMPLEX
Ag Ab
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TO BE CONTINUED ,
The free antigen and labelled antigen are separated and washed out .
The radioactivity of the labeled antigen antibody complex is measured
using scintillation counter or gamma counter.
The measured radioactivity is inversely proportional to the concentration of
antigen .
More is the concentration of antigen , lesser is the radioactivity of the
complex and vice versa .
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CALIBRATION CURVE :
 From these data, a standard binding curve, like the one shown in red, can be drawn.
 The samples to be assayed (the unknowns) are run in parallel .
 After determining the ratio of bound to free antigen in each unknown, the antigen
concentrations can be read directly from the standard curve.
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STEP FOLLOWED IN
RADIOIMMUNOASSAY
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PREPARATION AND CHARACTERIZATION OF ANTIGEN
 The antigen whose concentration is to be
measured , should be available in pure form.
 Also required to create antibodies ,in assay
process to form complex .
RADIO LABELING OF ANTIGEN
 TAGGING , where antigen is tagged with
radio active elements like 125I , 14C
PREPARATION OF SPECIFIC ANTIBODY
 Specific antibodies are produced by injecting the antigen in
mouse , rabbit, guinea pig & collect & purify the antibodies .
 In case of drug or other compounds (Eg . morphine ,
hormones ) which are not antigenic .
 They combined with albumin , to become antigenic .
DEVELOPMENT OF ASSAY
VALIDATION OF METHOD Parameter like sensitivity ,specificity,
precision, linearity etc
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PROCEDURE INVOLVED IN
RADIO IMMUNOASSAY
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THE ASSAY IS CARRIED OUT IN WELL PLATES (TYPICALLY 96 WELL PLATE)
 Where inner surface is coated with antibodies
RADIO ACTIVE ANTIGEN IS ADDED TO ALL THESE WELLS
 Which forms a complex with antibodies
THE UNLABELED ANTIGEN OF KNOWN CONCENTRATION AND ANTIGEN OF
UNKNOWN CONCENTRATION (SAMPLE)
 Where known conc. Added to multiple wells and
unknown conc. Added to one of the well
COMPETITION BETWEEN LABELED AND UNLABELED ANTIGEN TAKE PLACE
 Due to limited binding sites present in antibodies
RADIO LABELED ANTIGEN BOUNDED TO ANTIBODY
 Separated by precipitation/chromatography/gel
filtration
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ANTIBODY COMPLEX FORMED
• This leads to displacement of radio labeled antigen by
unlabeled antigen
AFTER INCUBATION TIME THE PLATE ARE REMOVED AND WASHED
• This remove free antigen and unbound
antigen
GAMMA OR SCINTILLATION COUNTER
• Measuring the radio activity
MORE CONC.OF FREE ANTIGEN LESSER RADIO ACTIVITY IN COMPLEX
FROM THE GRAPH OF RADIO ACTIVITY VS CONC.THE UNKNOWN ,CONC.OF ANTIGEN CAN
BE DETERMINED
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7/25/2021

Figure illustrates how a limited amount of antibody binding sites contained in a test tube or
microplate well can either bind unlabeled ligand (in this example, Hepatitis B Surface Antigen) or
radiolabeled ligand. As the amount of unlabeled ligand increases, there is consequently less
radiolabeled ligand bound. The unlabeled ligand can come from either a “calibration standard”
or the sample that you are trying to measure.
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SEPARATION TECHNIQUE
To separate free from bound labelled antigen separation techniques are :
a ) physical methods:-filtration ,chromatography ,electrophoresis, charcoal
dextran adsorption etc.
b) Chemical methods:-organic solvents such as ethanol, dioxane , PEG,
ammonium sulfate for precipitating antibody .
C) Immunological precipitation :- 2nd antibody
d)Solid phase systems :- with antibodies on solid support or matrix
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INSTRUMENTATION
The following steps are involved in RIA:
 Centrifuge
 Radioactive counter
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swing- bucket rotor
CENTRIFUGE
Fixed angle head rotor
generating 1200-2500
rpm
generating 3500-4000
rpm
pellet is formed at an
angle
pellet is formed at the
bottom of the test tube
The latter type is
preferred
The former type is
preferred
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RADIOACTIVE
COUNTER
Gamma
counters
Scintillation
Counters
gamma-energy emitting
isotopes, for instance : 125I-
the more common iodine-
isotope.
beta-energy-emitting
isotopes, such as :
tritium 3H and 14C-(Carbon-
14) isotopes.
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GAMMA COUNTER scintillation
Counter 7/25/2021

RIA OF DIGITALIS
Digitalis Commonly known as Foxglove leaves,
Generic name – Digitalis purpurea
Family - Scrophulariaceae.
It is also used for drug preparations that contain cardiac glycosides, like
Digoxin & Digitoxin extracted from various parts of the plant.
USES : Increase cardiac contractility
Antiarrhythmic agent
Atrial flutter
Atrial fibrillation
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PRINCIPLE OF RIA FOR DIGITALIS
The assay is based on the use of 125-iodine-labelled digoxin and of a gel equilibration technique
for the separation of antibody- bound and free digoxin.
Digoxin in serum samples competes with radio-labelled (125-1) digoxin derivative for binding sites
on the antibody to digoxin.
The unbound digoxin is then separated from bound form.
It is then quantified by counting radioactivity & concentration of unlabeled digoxin in serum
sample is calculated by comparison to digoxin standard .
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 Digoxin Standards - From 0.5- 8.0 ng/ml
 Anti-serum and Tracer solutions
 Digoxin antibodies raised in rabbits by subcutaneous
 Injections of digoxin-bovine serum albumin
 Phosphate saline buffer
 Digoxin Radio Immuno assay kit, with 3-0-succinyl
 Digoxigenin tyrosine 125-1.
Materials Used For Assay
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The assay is carried out using 3-O-succinvl digoxigenin tyrosine 125-Ⅰ.
1 ml of phosphate buffer solution + 0-50 µl of standard digoxin solution
+ 10 µl of 3-0-succinyl digoxigenin tyrosine 125-Ⅰ
Add 10 µl of digoxin anti-serum, all contents are mixed well.
Standard
Curve
PROCEDURE FOR ASSAY
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To 50µl of patient's plasma, + 1 ml of phosphate buffered saline.
To this add 10µ of labelled solution, & 10µl of digoxin anti-serum,
All contents are mixed well
All the tubes are allowed to stand and 0.5ml of charcoal solution is added to all tubes.
The tubes are then centrifuged, gamma radioactivity.
Procedure For
Unknowns
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RIA measurements of insulin
 RIA measurements of insulin combined with glucose estimations.
 Can diagnose hyperinsulinism due to insulin secreting tumors of the pancreas.
 Insulin (in micro units per ml) to glucose (in mg/100 ml) ratios of more than 0.30 is indicative
of this condition.
 Measurement of insulin and C-peptide concentrations following arginine stimulation Test .
 In this test, 300 ml of a 10% solution of arginine is infused into a fasting patient who has not
had insulin, over a period of 30 minutes and blood taken at - 0, 15, 30, 45, 60 and 90
minutes.
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 In Type I, insulin dependent diabetes, basal levels of C-peptide would be less than 0.3 ng/ml
and there would be no response to arginine.
 If basal C-peptide is low, say 0.3-1.0 ng/ml, and there is some response to arginine (a 100%
rise), the patient is still insulin deficient.
 In typical Type II diabetes, basal levels would be 2.5 ng/ml or over, and a 100% rise on
stimulation would be seen.
 RIA for microalbuminuria is becoming popular as a predictor of the development of clinical
nephropathy in diabetics.
TO BE CONTINUED ,
7/25/2021

MERITS OF RADIOIMMUNOASSAY
 High specificity and hence there is no interference from other substances.
 High sensitivity – Immuno reaction are very sensitive , so when antibody with high
affinity are used , it is possible to detect antigen up to a few picogram (10-12 g).
 High precision and accuracy
 Applicable to wide variety of compounds in various fields
pharmacology
Endocrinology
Oncology
Epidemiology
Clinical immunology
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DEMERITS OF RADIOIMMUNOASSAY
 Expensive compared to other methods ,as radioactive materials are used .
 Radiation hazards because of Usage of 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.
 Development of specific antibodies to the antigen ..
 Compliant technique than other methods compared to Tlc , Hptlc , etc.. 7/25/2021
KMCH COLLEGE OF PHARMA
38

APPLICATION OF RADIO IMMUNOASSAY
1.The test can be used to determine very small quantities (e.g. nanogram) of antigens and antibodies
in the serum.
2.The test is used for quantitation of hormones, drugs(Digitoxin or digoxin ), HBsAg in blood ,HIV 1& 2
Virus and other viral antigens.
3.Analyze nanomolar and picomolar concentrations of hormones in biological fluids.
4.Certain abused drugs , Eg . MORPHINE
5.Ria has been used to assay plasma levels of :
1.Most of hormones
2.Insulin in human plasma
3.Beta - HCG in females
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DRAWBACKS :
 The cost of equipment and reagents are expensive .
 Short shelf life of radiolabeled compounds .
 The problems associated with the disposal of radioactive waste.
 Hazards if preparing and handling the radioactive antigen.
 Both 125I or 131I emit gamma radiation that requires special
counting equipment . 7/25/2021
40

THE SPECIFICITY OF AN RIA IS IMPROVED BY:
(a) The use of pure immunogens or conjugates.
(b) Pure labelled compounds.
(c) Pure analyte for assay, pre-extracted and purified if needed.
(d) Immunization with specific subunits, e.g. b-hCG
(e) Saturation with cross reactants, e.g. TSH antibody with hCG
(f) Use of monoclonal antibodies of high specificity 7/25/2021
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7/25/2021

REFERENCE
 https://microbenotes.com/radioimmunoassay-principle-uses-and-limitations/
 https://www.google.com/search?q=radio+immunoassay&oq=&aqs=chrome.2.69i59i450l8
.1354187j0j7&sourceid=chrome&ie=UTF-8#
 Text book of instrumental analysis by the author Ravi shanker
 https://www.brainkart.com/article/Radioimmunoassay--Instrumentation_30954/
 The production of antibodies for radioimmunoassay ;
https://pubmed.ncbi.nlm.nih.gov/772533
 Journal of Perkin elmerhttps://www.perkinelmer.com/lab-products-and-
services/application-support-knowledgebase/radiometric/radioimmunoassays.html
 https://www.amjmed.com/article/0002-9343(71)90237-3/fulltext
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RADIO IMMUNO ASSAY

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