Radio Immuno Assay

Prepared By
Dr.J.Swaminathan M.Pharm.,MBA., Ph.D.
Associate Professor,
ADHIPARASAKTHI COLLEGE OF PHARMACY,
MELMARUVATHUR – 603 319
RIA
( Radio Immuno Assay)
RADIO IMMUNO ASSAY -
DR.J.SAMINATHAN, APCP, MLMR 1

RADIO IMMUNO ASSAY - DR.J.SAMINATHAN,
APCP, MLMR
3

Definition
Introduction
Principle and Theory
Methods in RIA
Other Immuno Assays
Application
Reference

Radioimmunoassay (RIA) is a scientific method used to test antigens (for
example, hormone levels in the blood) without the need to use a bioassays.
Radioimmunoassay (RIA) is a Radio-analytical technique with remarkable
sensitivity and a high degree of specificity that is widely used for the estimation
of a variety of molecules present in complex matrices. Also known as Radio
tracer technique and best example of invitro diagnosis technique using radio
isotopes.
This technique is used over a wide spectra of substances such as hormones,
steroids, vitamins, drugs, tumor markers and viral antigens.
Radio Immuno Assay
Use of radio
active material
Antigen antibody
binding theory
Detection of
compoundRADIO IMMUNO ASSAY -

This isotopic measuring method was developed in 1959 by two Americans,
biophysicist Rosalyn Yalow and physician Solomon A. Berson.
RIA combines the specificity of an antigen-antibody reaction with sensitivity of
radioactivity measurements.
This is a technique used for detection of micro quantities of protein, viral antigens,
antibodies, structural proteins, vitamins and drug and their metabolites.
It can also be used for detection of pictogram quantities (10−12 g) of biological
constituents present in biological fluid.
RIA is used in place of bioassay in various branches of science like Biochemistry,
Microbiology, and Hematology and Clinical pharmacology.

RIA works on basic principle of biochemistry that competitive binding between
antigens for same antibody binding site.
The competition of an analyte with its radioisotopically labeled counterpart for a
limited amount of antibody, the specific reagent, is the underlying principle of this
technique. Increasing the analyte concentration inhibits the binding of the labeled
analyte to the antibody.
Ag + Ag* + Ab  AgAb + Ag*Ab + 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*: radio labelled ligand

A
B
C
Antibody
Unlabeled antigen
Labeled antigen
Now how the competition occur with increase the concentration of unlabeled
antigen in the system of RIA in three different cases A, B and C.
Here first antibodies bound with labeled antigen are put into the known
concentration of analyte solution and it is observed how the labeled antigen free
from antibody and unlabeled will bind in place of there.

The concentration of the unknown analyte is thus obtained by comparing its
inhibitory Effect on the binding of the labeled analyte to that of a known standard.
After determining the ratio of bound to free antigen in each unknown, the antigen
concentrations can be read directly from the standard curve (as shown above
After determining the ratio of bound to free antigen in each unknown, the antigen
concentrations can be read directly from the standard curve (as shown above).
1
2
1. – Ratio in unknown and
2. - Antigen in unknown
Red line – binding line
Green line – free labeled antigen
RADIO IMMUNO ASSAY - DR.J.SAMINATHAN, APCP, MLMR12

From graph we can also calculate %F (fraction of free labeled antigen) and %B
(fraction of bound labeled antigen).
&
• F – amount of free labeled antigen
• B - amount of bound labeled antigen
Here as the concentration of unlabeled increase it replaces the labeled bound
antigen by competitive binding it inhibiting the binding of labeled one.
Antigen- antibody complex depend on antibody affinity (Ka) –
The affinity with which antibody binds antigen results from a balance between
the attractive and repulsive forcesRADIO IMMUNO ASSAY -

Advantages
Highly specific: Immune reactions are specific,the greater the specificity of the
antiserum, the greater the specificity of the assay.
High sensitivity : Immune reactions are sensitive, Using antibodies of high
affinity it is possible to detect a few picograms (10−12 g) of antigen in the tube.
Accuracy and Precision
Disadvantages
Radiation hazards: Uses 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 the
Assay System
Development of the Assay System
1. A crucial step is separation of unbound antigens
2. This achieved by binding the antibodies to the microtitre well surface
[Solid phase RIA]
3. Antigens bound to the fixed antibodies remain stuck to the inner surface
4. Decanting & washing the well removes unbound antigens
5. Other techniques of separation: Centrifugation

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

1. Radio labelling of the Antigen or radio labelled production
2. Preparation & characterisation of the Antigen [Ligand to be
analysed]
3. Preparation of the Specific Antibody
4. Development of Assay System or separation techniques
Methods in RIA :

1. Preparation and radio labeling of antigen
Antigen preparation:
 Synthesis of the molecule
 Isolation from natural sources
Radiolabelling [Tagging procedure] :
 Two most commonly used radio labels in RIA 3 H and 125 I although Se, P, Co
14 C and 131 I have also been used. but these have some limitations that are –
 32P and 57Co = limited by stereo chemical aspects because drug
naturally contain phosphorous and cobalt
 14 C = low specific activity field
 131 I = Short decay half life and radio degradation character
on the other hand 3 H and 125 I have advantages as follows:
 3 H = Direct incorporate into molecular structure, half lives(12 years)
 125 I= High activity and ease of counting (half lives 60 days)
75 32 57

Technique of labeling of drug or antigen with 3H and 125I:
Specific tritium labels are generally obtained by reducing an appropriate
precursor in presence of 3H.
Labeling of drug with 125I include chloramines-T, monochloride exchange
and enzymatic iodination methods. The most suitable iodination
procedure is depending on the stability of the drug and the specific activity
that is sufficient to meet the sensitivity requirements of assy.
Comparison of 3H and 125I is as follow:
Sr no. Tritium (3H) Iodine (125I)
1 It is more efficient when relatively small
numbers of samples are assayed.
It is more efficient as the numbers of samples are
increased.
2
Such type of problem does not occur.
It’s having quality control problems such as
damage during the reaction and radiation damage
following synthesis.
3 Having advantages of long half life, higher
affinity.
It has short half life dictates frequent preparation.

it is possible to find the specific antibody for drug(antigen) as a result of
sensitivity reaction
Several drugs which have induced antibodies production due their inherent
antigenicity like – penicillin, strychnine, tetracycline, sulphonamide and
procainamide. However their low specificity and limited availability makes their
use rather improbable.
In most cases the drugs (analysed antigen) are bind with suitable carrier protein
to make conjugated antigen immunogenic.
There are several reactive groups on protein carrier which can used for the
purpose of conjugation of standard drug(antigen).these groups include the
terminal amino and carboxyl groups, ε-amino group of lysine, the carboxyl group
of aspartic and glutamic acid, the phenolic group of tyrosine.
2. Preparation purification of drug-protein conjugate(ligand-antigen) to be
analyzed:

The most commonly used methods for conjugation are as follows:
Carbodimide and glutaraldehyde reaction
Carbony-diimidazole reaction
Scotten-baumann reaction
Mannich reaction
Diazotization reaction
The method chosen for conjugated will depend on the functional groups
available for coupling the drug to protein and no. of drugs molecules
which are to be coupled to protein.

 Preparation :
 Once the pure antigen prepared then it is emulsified into equal volumes of
saline and Freud’s adjuvant (contain alum ppts, natural detergents, mineral oils,
killed mycobacterium) to get final concentration of 10 to 50mg/ml.
 One ml of the emulsion is injected intradermally, subcutaneously, and/or
intramuscularly at weekly or monthly intervals into multiple sites of a suitable
animal species such as rat, guinea pigs or rabbits. A suitable animal species is
usually dictated by the size of the animal facilities.
 Animals are tested after 3-4 weeks and then blood is collected and separated.
The resulting blood containing antibody called antiserum. It is directly used in
assay and should be stored at 4°c.
 Characterization:
 Characterization of antiserum done by fractionation, immunoadsorption or
immunosaturation technique.
3. Preparation & characterization of the Specific and high affinity Antibodies:

4. Development of suitable separation techniques to separate free from
bound standard drug:
 Several methods that employ physiochemical and immunological separation have been
devised as follows –
Physical methods: Filtration, Chromatography, Electrophoresis, charcoal- dextran
adsorption, and ion exchange resin.
Disadvantages: it is tend to be time dependent and harsh so they may remove bound drug
from antibody during separation.
Chemical method: organic solvents such as ethanol, dioxane and polyethylene glycol
(PEG), and salts such as sodium, zinc, ammonium sulfate.
Disadvantages: chemical precipitation may precipitate free as well as bound drug during
separation depending on physicochemical nature of drug.
Second antibody method: it is most physiologic procedure to precipitate bound antigen.
This method employs, an antibody against gamma globulin of the animal species used to
produce antidrug antibody to the plasma serum to precipitate drug-antibody complex.
Disadvantages: This technique have required prolong incubation time.

Other related Immuno Assay:
Related methods include the use of antibodies to specifically bind antigenic
substances.
They do not include competitive protein binding assays (CPBA) or receptor site
binding assay (RSBA).
The most commonly related method is Immunoradiometric (IRM) – the
technique which employs an excess of labeled antibody to quantify the drug in the
system. The principle of the system displayed in figure as below.
S + Ab‫٭‬ SAb‫٭‬ + Ab‫٭‬ S Ab‫٭‬ + SPAb‫٭‬
SP
S is unknown or standard antigen, Ab‫٭‬ is labeled antibody, and SP is the antigen
coupled to a solid support.
SAb‫٭‬ fraction will remain in the supernatant, where as the SPAb‫٭‬ will be coupled
to the solid support.

•Advantages:
The antibody can be more readily labeled with high specific activity using 125I.
No need of any specific separation technique as in RIA.
Instead of using radio tracers several other methods rely on alternate labeling
procedures. They include hemaglutination-inhibition, fluorescence, and free radical
or spin label and last Enzyme Immuno-Assays.
Only Enzyme Immuno-Assays (EIA) – has gained wide acceptance. The
theoretical concept of EIA is same as RIA, principle of EIA is as follows:
P S‫+٭‬ Ab S‫٭‬Ab
+
S SAb
S and S‫٭‬ are pure and enzyme linked
drug, respectively; Ab is antibody; and P is
the measurable reaction product that can
result only when free S‫٭‬ react with an
appropriate antibody.

•Advantages of EIA over RIA:
•No radiation hazard
•Economy of quantitation
•Homogenous EIA does not require separation of free and bound ligands
(antigen)
•Disadvantages:
•Relatively insensitive due to the low catalytic activity of enzymes
•A linkage specificity problem when same linkage used for antibody conjugates
and enzyme conjugates.
Several drug EIA systems are currently in use in the clinical chemistry
laboratory, primarily for the detection of drugs of abuse and quantification of
Anticonvulsant and digoxin plasma concentration.

Application of RIA:
Analysis of hormones, vitamins, metabolites, diagnostic markers:
E.g. ACTH, FSH, triiodothyronine (T3) and thyroxine (T4), Glucagons, Insulin,
Testosterone, vitamin B12, prostaglandins, glucocorticoids,
Therapeutic drug monitoring:
Barbiturates, morphine, digoxin,
Diagnostic procedures for detecting infection :
HIV, Hepatitis A, B etc
Tumour markers:
• RIA of tumour markers such as alpha-fetoprotein (AFP), carcinoembrionic
antigen (CEA), b-HCG for choroid -carcinoma, prostate specific antigen (PSA) for
prostate cancer, are available for detection and management of cancer
Non clinical application: such as veterinary science, food processing industry,
drug industry, forensic science and environmental monitoring.

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