Radioimmunoassay is an immunoassay technique that uses antibodies and radioactive-labeled antigens to measure concentrations of antigens. It works by having the radioactive antigen compete with unlabeled antigen in a sample for binding to an antibody. The more unlabeled antigen that is present, the less radioactive antigen will bind to the antibody. By measuring the amount of radioactive antigen that is bound versus unbound, the concentration of the unlabeled antigen in the sample can be determined. Radioimmunoassay is a sensitive technique that can detect picogram levels of antigens and is useful for applications like cancer detection, hormone level measurements, and narcotic detection.

1. Radioimmunoassay
BY- ASST. PROF. SANJAYKUMAR UCHIBAGLE,
GHRU, SAIKHEDA.
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2. Immunoassay
 An immunoassay is test that uses Antibody & Antigen complex.
 An Antibody : Antigen Complex is also known as Immune-Complex.
 Immune refer to an immune response that cause the body to generate Antibodies
 Assay refer to the test.
 Immunoassay is refer to test that utilize immune complexing when Antibodies :
Antigen are brought together.
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3. Antibody, Antigen & Analyte
 An Antigen(Ag) is the substances that the body is trying to fight off by
mounting an immune response.
 An Antibody(Ab) is protein that is produce by the body in response of an
“invading” (foreign) substances.
 An Immunogen is a substances that elicits immune response.
(Drug-Protein Conjugate)
 An Analyte is anything measured by lab.
 In Immunoassay testing Analyte may be either an Antigen or antibody.
 In Immunoassay utilize one or more selected antibodies to detect the
analyte of interest.
 The analyte being measured may be
That are naturally present inside of the body.(T3 & T4 Hormone).
The body produces but not typically are not present ( Such as cancer
Antigen).
Do not naturally occurring inside of the body.
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4. Structure of Antibodies
 Ab are produce by the β-
lymphocytes.
 The most common one is
Immunoglobin G (IgG).
 IgG is protein composed of 2 main
structural and functional region.
 Fab Region: Contain the Ag
binding site that varies between
different Ab.
 FC region: Constant structure
within an Ab.
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5. History
 Developed in 1959 by Rosalyn
Yellow for measurement of
insulin in Plasma.
 It represented first time that
hormone level in blood could be
detected by an in vitro assay.
 In 1977 received the Nobel
Prize.
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6. 1. The amount of Ab per tube is kept constant, the amount of Ag is
add(known or unknown) is the variable parameter.
2. The added Ag will be distributed between a bound (b) and free (f) form
This distribution is governed by the association constant.
Principle of RIA
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7. Principle of RIA
→ Competitive binding of radiolabelled Ag & unlabelled Ag to high affinity Ab.
→ The labelled Ag is mixed with the Ab at a concentration that saturate the Ag-binding site of
Ab.
→ As the con. of the unlabelled Ag is ↑ more labelled Ag will be replaced from the Ab binding
site.
→ The decrease in the amount of radiolabelled Ag bound specific Ab in the presence of test
sample is measured to determine the amount of Ag is present in test samples.
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8. Principle of RIA
→ In the standard condition, amount of labelled Ag bound to the Ab decrease as the amount of
unlabelled Ag is increased is sample.
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9. Reagent used in RIA
→ A tracer i.e. a labelled ligand.
→ A binder (Ab) which is the specific antiserum.
→ A separation system to separate to separate the
bound and free phases.
→ A STANDARD in highly pure form.
→ A free human antiserum.
Ligand -an ion or molecule, which
donates a pair of electrons to the central
metal atom or ion to form a coordination
complex.
Antiserum- blood serum that contains specific
antibodies against an antigen (foreign agent), such as
an infective organism or poisonous substance
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10. Reagent used in RIA: Tracer
 → The radioisotope used are,
β emitters -3H, 14C
γ emitters -125 I
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11. Reagent used in RIA: Tracer
Tritium 3H
→Weak β ray emitter
→Significantly lower than 14 C
→Long physical half life of 12.3 yr
→Biological half life: 10-12 days
→Produced by neutron bombardment of lower hydrogen isotope.
→Used for the drugs like Protein and Amino acids.
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12. Reagent used in RIA: Tracer
Carbon 14:
→Weak β ray emitter.
→Long Physical Half Life (5.7 χ 10000 yr)
→Biological Half Life: 12 days, unbound 40
days.
→Commercially available as Barium
Carbonate 14 C
Iodine 125:
→Low γ emission: 35.4 keV.
→High Specific Activity.
→Short Physical Half-Life: 60 days.
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13. Reagent used in RIA: Tracer
Positron Vs Gamma Isotopes:
→The positron (β-emitting) radionuclides
are mainly restricted for in vitro exp.
→The γ emitting radionuclides are useful
for in vivo imaging.
Other Commonly Used Isotopes:
→Positron:11C, 13N, 15O, 18F.
→Gamma: 111Indium, 123 I, 131
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14. Reagent used in RIA: Tracer
125 I is most favoured because:
→It can be obtained with specific activity & almost 100% isotopes
abundance, thus reducing counting time and being economics.
→Convenient half life 60.2 days hence shelf life for labelled Ag is
long.
→Iodine is natural constituent of Thyroxine & TriidoThyronine.
→It can be easily introduce into peptide molecules, steroids.
→γ Emission permit the use of simple inexpensive equipment for
counting radioactivity.
Disadvantages:
→Damage may be occur to
the ligand during storages.
→Health Hazards More.
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15. Reagent used in RIA: Tracer
3 H is more efficient when
smallsample to be determined:
Advantages:
→Long Shelf Life 12.3 yr.
→Highly affinity & no requirements of
derivative preparations.
→Minimal Health Hazards.
Disadvantages:
→Required Scintillation Counter Which Is Costly
→ Low Specific Activity.
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16. Specific Antiserum(Binder/Ab)
→It is prepared by injecting (repeatedly) the Ag together with Freund’s Adjuvants into
suitable animals such as guinea pig, rabbit or goat.
→Molecules like thyroid hormone, steroids, drugs are not immunogenic. So they are
conjugated to carrier proteins and polymers make them immunogenic.
Freund’s Adjuvants : irreplaceable components of induction protocols of many
experimental animal models of autoimmune disease
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17. Separation Systems.
→It is required because the bound fraction does not ppt spontaneously at low conc.
→Variety of procedures are available.
1. Physical: Filtration, Chromatography, Electrophoresis, Charcoal-Dextan
adsorption.
2. Chemical : Ethanol, Dioxane, PEG, Zinc & Ammonium Sulphate.
3. Solid Phase Systems :
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18. Standard in Highly Pure Forms
→ Drugs, Protein & Hormones must be in pure forms so they can be
diluted.
→ Standards are prepared in Ligand free serum.
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19. Ligand Free Human Srum
→ It is prepared by treating human serum with Charcoal.
→It can also be prepared by collecting serum from volunteers in whom of that
ligand or Hormone has been inhibiting by treating with the appropriate
drugs.
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20. General Procedure for
Preparing RIA Analysis
→ A known quantity of an Ag is made radioactive.
→ A radiolabelled Ag is mixed with known amount Ab for that Ag & as a
result, two chemically bind to one another.
→ A sample of serum from the patient containing an unknown quantity of
that same Ag is added.
→ This caused unlabelled Ag from the serum to compete with RADIO
labelled Ag for Ab binding site.
→ As the con. of Unlabelled Ag increase, more of it bind to the Ab.
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21. General Procedure for
Preparing RIA Analysis
→ And by displacing the radio labelled variant and reduce the ratio of Ab-
bound radio labelled Ag to free radio labelled Ag.
→ The bound Ag are the separated from the unbound form.
→ The radioactivity of the free Ag remain in the supernatant is measured.
→ Separating bound unbound Ag is crucial.
→Initially, the method of separation employed was the use of second “Anti-
Antibody”.
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23. Advantages of RIA
→ RIA is very sensitive techniques used to measured the conc. of Ag
up to pictogram levels.
→ It is structurally specific Ag : Ab reaction are highly specific.
→ It is indirect method of analysis.
→It is saturation analysis as active reagents added in smaller
quantity than that of the analyte.
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24. Dis-advantages of RIA
→ Prolonged Reaction Time.
→ Highly dil. Regent Required
→ Radioactive Iodine Is Not Cheap Reagents.
→ Possibility of Health Hazards Due to Handling of Radioisotopes.
→ All The Reagents Must Be in The Pure Forms only.
→ Limited Assay Ranges.
→ Lack of Direct Relationship Linear Standards Response Curve.
→ Difficulties of Automation.
→ Time Consuming.
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25. Uses of RIA
→ Narcotic Detection.
→ Blood Bank Screening Of Hepatitis.
→ Early Cancer Detection.
→ Measurements of Growth Hormone Level.
→ Tracking of Leukaemia Virus.
→ Diagnosis and Treatment of Peptide Ulcers.
→ Study of The Neuro-transmitters.
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26. Recent Application of RIA
→ Estradiol measurement in Estradiol studies of breast cancer.
→ Evaluation of prostate specific antigen n prostate cancer protein and in non
cancer patients and in non cancerous prostatic disease patients.
→ Evaluation of enzyme immunoassay and radioimmunoassay methods foe
the measurements of plasma oxytocin.
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27. Any Questions?
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28. Thank You!
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