Radioimmunoassay
BY- ASST. PROF. SANJAYKUMAR UCHIBAGLE,
GHRU, SAIKHEDA.
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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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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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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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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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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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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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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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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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Reagent used in RIA: Tracer
 → The radioisotope used are,
β emitters -3H, 14C
γ emitters -125 I
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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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Any Questions?
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Thank You!
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