Radioimmunoassay

Presented by :
Justin K Joy
First Year M.Pharm Pharmaceutical Analysis
St.James college of Pharmaceutical Sciences.
 Immunoassay
• An immunoassay is a test that uses antibody and antigen
complexes
• An antibody: antigen complex is also known as an immune-
complex
• “Immuno” refers to an immune response that causes the body
to generate antibodies
• “assay” refers to a test
• immunoassay is a test that utilizes immuno complexing when
antibodies and antigens are brought together

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 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.

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 Antibodies, Antigens and Analytes
 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)

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 Structure of Antibodies
• Antibodies are produced by the B lymphocytes.
• The most common 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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 Radioimmunoassay
 Radio Immuno Assay (RIA) is an elegant tech. in analytical
chemistry.

 If substance to be analysed 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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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

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 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]

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 Principle Of RIA
Competitive binding of radiolabelled antigen and
unlabelled antigen to a high affinity antibody.

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.

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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 std Condition, amount of labelled antigen bound to the antibody decreases as

the amount of unlabelled antigen increases in sample.

Antibody

Unlabeled antigen

Labeled antigen

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 Reagents used in RIA:
1. A tracer i.e. a labelled 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.

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 Reagents used in RIA: Tracer

The radioisotopes used are –

Beta emitters- 3H and 14C

Gamma emitters- 125I

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 Reagents used in RIA: Tracer
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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 Reagents used in RIA: Tracer

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 carbonate-14C

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 Reagents used in RIA: Tracer
Iodine-125:
 Low γ−emission: 35.4 keV
 High specific activity
 Short physical half-life: 60 days

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 Reagents used in RIA: Tracer
 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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 Reagents used in RIA: Tracer
 125I is most favoured because-
• 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
labelled antigen is long.
• Iodine is natural constituent of thyroxine and tri-
iodothyronine.
• 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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 Reagents used in RIA: Tracer

 Disadvantages-
o The damage to the ligand may occur during storage.
o Health hazards are more.

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 Reagents used in RIA: Tracer
3H is more efficient when relatively small sample is
to be determined.
 Advantages-
• Long shelf life (12.3 yrs).
• Higher affinity and no necessity for derivative preparation.
• Minimal health hazards.
 Disadvantages-
• Requires scintillation counter which is costly
• Low specific activity (10000 14C = 100 3H = 1 125I).

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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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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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A STANDARD ie 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.

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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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General Procedure for Performing a RIA
Analysis
• A known quantity of an antigen is made radioactive
• This radiolabeled 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 radiolabeled antigen for
antibody binding sites
• As the concentration of "cold" antigen is increase, more of
it binds to the antibody.
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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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 Advantages Of RIA
 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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 Disadvantages Of RIA
• Prolonged reaction time (in days) as a consequence highly
diluted reagent is used.
• Radioactive Iodine used in is not a cheap reagent.
• Possible health hazards due to handling of radioisotopes.
• All the reagents must be added precisely.
• Limited assay range.
• Lack of direct linear relationship between analyte
concentration and signal response.
• Difficulty of automation.
• Lengthy counting time.
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 Uses for RIA
 Narcotics (drug) detection,
 Blood bank screening for the hepatitis (a highly
contagious condition) virus,
 Early cancer detection,
 Measurement of growth hormone levels,
 Tracking of the leukemia virus,
 Diagnosis and treatment of peptic ulcers, and
 Research with brain chemicals called neurotransmitters

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Recent applications….
Refinement of Zalcitabine pharmaco kinetics.

Estradiol measurement in translational studies of breast cancer.

Plasma estrogen measurement with use of radioimmunoassay has been instrumental in
the development of aromatase inhibitors for endocrine therapy of postmenopausal
breast cancer

Significance of prostate specific antigen in prostate cancer

patients and in non cancerous prostatic disease patients.

Evaluation of enzyme immunoassay and radioimmunoassay

methods for the measurement of plasma oxytocin

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INSTRUMENTATION
• Instrument name: Gamma Counter
(WIZARD2)
•

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 References
Lanning PE. Estradiol measurement in transitional studies of breast cancer. Elsevier
2015;99:26-31. Available from: URL:http://www.elsevier.com/locate/steroids.

Muhammed H, Parveen Z, Shahid K, Samreen I, Ajasrasool s. Significance of prostate specific

antigen in prostate cancer patients and in non cancerous prostatic disease patients. Journal of
Pakistan medical association 2007;57:248-51.

John MA, Mark JS, Ross GH, Gene DM, Thaddeus HG et.al. Zalcitabine population
pharmacokinetics: application of radioimmunoassay. American society for microbiology
1998;42(2):409-13. Available from: URL:http://aac.asm.org.

Angela S, Philip MM, Daniel AN, Benjamin A, Tabak MA et.al. Evaluation of enzyme
immunoassay and radioimmunoassay method for the measurement of plasma oxytocin. National
institute of health, physchosom med 2011 june;73(5):393-400.

SabahA.Presentation on radioimmunoassay. Department of pharmacology. Newdelhi

URL:http://www.slideshare.com/radioimmunoassay.

Yalow R, Berson S. Immunoassay of endogenous plasma insulin in man. J. Clin. Invest 1960;
39: 1157-75.
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 THANK
YOU……….

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