Radioimmuno assay/instruments/elements used/application

1. Sam Higginbottom University of Agriculture
Technology and Sciences Prayagraj, India
Radioimmune Assay
Submitted to:
Prof. (Dr). P.malai rajan
(Hod, Department of Pharmaceutical Sciences, SIHAS, SHUATS,
Prayagraj, U.P, India)
Date of Submission: 08/03/2021
Submitted By:
Name: Imdad H. Mukeri
Bachelor of Pharmacy (8th sem)
Id No. 17BPH084
Subject: Advance Instrumentation technique (801T)
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2. Contents
• History and Introduction Of RIA
• Importance And Various components of RIA
• Principle of RIA And its Reagents used
• Method of separation and its procedure
• Limitation and its Application
• Conclusion And References
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3. History
• Developed in 1959 by
Rosalyn Yalow and Solomon
Berson for measurement of
insulin in plasma.
• 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 Rosalyn yalow
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4. Immunoassay
• An immunoassay is a test that uses antibody and
antigen complexes
• An antibody: antigen complex is also known as an
immune complex
• “Immune” refers to an immune response that
causes the body to generate antibodies
• “Assay” refers to a test
• Immunoassay is a test that utilizes immune
complexing when antibodies and antigens are
brought together
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5. Antibodies, Antigens and Analytes
• An antibody is a protein that is produced by the body in
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, the drug is the antigen that binds to the
antibody.
• An immunogen is a substance that elicits immune response.
E.g. drug-protein conjugate.
• An analyte is anything measured by a laboratory test.
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6. • 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)
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7. Radioimmunoassay
• Radio Immuno Assay (RIA) is an elegant tech. in analytical
chemistry.
• If substance to be analyzed is in very low quantities, in the
orders of micrograms, nanograms, conventional methods like
gravimetric and colorimetric method fail.
• RIA finds extensive application in the assay of many
substances which are present in trace amount in blood.
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8. Importance of radio immune assay
• The investigation was conducted to study the role of the
determination of tumor markers (CEA, β2-microglobulin, IgE
and ferritin) in patients with malignant lymphomas.
• Radio immuno assay is very sensitive technique used to
measure concentrations of antigen without the need to use a
bioassay. It can measure one trillionth (10-12) of a gram of
material per milliliter of blood.
• It is structurally specific as antigen: antibody reaction are
highly specific.
• It is indirect method of analysis.
• It is a saturation analysis as active reagent added in smaller
quantity than that of analyte.
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9. Principle of RIA
• The amount of Ab per tube is kept constant, the amount of
antigen added (known or unknown) is the variable parameter.
• The added antigen will be distributed between a bound (B)
and a free (F) fraction.
• This distribution is governed by the association constant (KA )
of the Ab:
Ab + Ag AgAb
K = [AbAg] /[Ab][Ag]
• Competitive binding of radiolabelled antigen and unlabelled
antigen to a high affinity antibody.
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10. Principle of RIA
• The labelled antigen is mixed with the antibody at a
concentration that saturates the antigen –binding sites of the
antibody.
• As the concentration of the unlabelled antigen increases more
labelled antigen will be replaced from the binding site.
• The decrease in the amount of radiolabelled antigen bound
specific antibody in the presence of the test samples is
measured to determine the amount of antigen Present in the
test sample.
• In Standard Condition, amount of labelled antigen bound to
the antibody decreases as the amount of unlabelled antigen
increases in sample.
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11. Reagents used in RIA
1. A tracer i.e. a labeled ligand.
2. A binder (Antibody) which is the specific antiserum.
3. A separation system to separate to separate the ‘bound’ and
‘free’ phases.
4. A standard (in highly pure form)
5. A free human antiserum.
The radioisotopes used are
 Beta emitters3H and 14C
 Gamma emitters125I
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12. Reagents used in RIA
Tritium – 3H:
• Weak β− ray emitter
• Significantly lower energy than 14C
• Long physical half-life of 12.3 yrs
• Biological half-life: 10 – 12 days
• Produced by neutron bombardment of a lower hydrogen
isotope
• Used for drugs like proteins and amino acids
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13. Reagents used in RIA
Carbon-14:
• Weak β− ray emitter
• Long physical half-life (~ 5.7 x 103 yrs)
• Biological half-life: Bound – 12days; Unbound – 40days
• Commercially available as Barium carbonate14C
Iodine-125:
• Low γ−emission: 35.4 keV
• High specific activity
• Short physical half-life: 60 days
It can be obtained with specific activity and
almost 100% isotopic abundance, thus reducing
counting time and being economic. • Convenient
half-life (60.2 days) hence shelf life for labeled
antigen is long. • Iodine is natural constituent of
thyroxin and triiodothyronine. • It can be easily
introduced into peptide molecules, steroids. •
Gamma emission permits the use of simple
inexpensive equipment for counting radioactivity.
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14. Reagents used in RIA
Positron vs. Gamma Isotopes:
• The positron (β-emitting) radionuclides are mainly restricted
for in-vitro experiments.
• The γ-emitting radionuclides are useful for in-vivo imaging
Other commonly used isotopes:
• Positron: 11C, 13N, 15O, and 18F.
• Gamma : 111In(indium), 123I, 131I, 153Sm(Samarium), 75Se.
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15. Specific antiserum(binder/antibody)
• It is prepared by injecting (repeatedly) the antigen together
with Freund’s adjuvant into suitable animal such as guinea
pig, rabbit or goat.
• Molecule like thyroid hormone, steroids, drug are not
immunogenic. So they are conjugated to carrier proteins and
polymer to make them immunogenic
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16. SEPERATION SYSTEM
It is required because the bound fraction does not precipitate
spontaneously at the low concentration. Variety of procedure
are available
a)Physical method
Filtration,chromatography,electrophoresis,charcoal dextran
adsorption
b)Chemical method
Organic solvent such as ethanol,dioxane,PEG or salts such as
sodium ,zinc and ammonium sulphate.
C)Solid phase system
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17. A STANDARD i.e. LIGAND (ANALYTE) IN HIGHLY PURE
FORM
• Drugs , protein, hormone etc must be in pure form so they
can be diluted .
• Standard are prepared in ligand free serum.
• In case of protein ,hormones the standard should be prepared
preferably with the hormone from the species from which the
serum is going to be analysed.
LIGAND FREE HUMAN SERUM
• It is prepared by treating human serum with charcoal .
• It can also be prepared by collecting serum from volunteers in
whom production of that ligand or hormone has been
inhibited by treatment with an appropriate drug
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18. General Procedure for Performing a
RIA Analysis
• A known quantity of an antigen is made radioactive
• This radio labeled antigen is then mixed with a known amount
of antibody for that antigen, and as a result, the two
chemically bind to one another.
• A sample of serum from a patient containing an unknown
quantity of that same antigen is added
• This causes the unlabeled (or "cold") antigen from the serum
to compete with the radio labeled antigen for antibody
binding sites
• As the concentration of "cold" antigen is increase, more of it
binds to the antibody
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19. General Procedure for Performing a
RIA Analysis
• And by displacing the radio labelled variant and reduces the
ratio of antibody-bound radio labelled antigen to free radio
labelled antigen.
• The bound antigens are then separated from the unbound
ones
• The radioactivity of the free antigen remaining in the
supernatant is measured.
• Separating bound from unbound antigen is crucial
• Initially, the method of separation employed was the use of a
second "anti-antibody“.
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21. The limitations of the RIA include:
• The main drawback of RIA are the expensive and hazards of
preparing and handling the Radioactive antigen.
• The gamma emitting isotope such as I125 Requires special
counting equipment.
• Require special license to handle Radioactive materials.
• The cost of equipment and reagents
• Short shelf-life of radiolabeled compounds
• The problems associated with the disposal of radioactive
waste.
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22. Application of Radio Immuno Assay
1. The test can be used to determine very small quantities (e.g.
monogram) of antigens and antibodies in the serum.
2. The test is used for quantitation of hormones, drugs, HBsAg,
and other viral antigens.
3. Analyze nanomolar and picomolar concentrations of
hormones in biological fluids
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23. Conclusion
In above we study about Radio Immuno Assay (RIA) is an
elegant tech. in analytical chemistry. RIA used to be analyzed
is in very low quantities, in the orders of micrograms,
nanograms.
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24. References
• https://microbenotes.com/radioimmunoassay-principle-uses-
and-limitations/ (accessed on 07/03/2021)
• https://www.slideshare.net/justinsolin/radioimmunoassay-
56112157 (accessed on 06/03/2021)
• http://mcb.berkeley.edu/courses/mcb150/lecture5/Lecture5(
6).pdf (accessed on 06/03/2021)
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25. Thank you
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