CALCUTTA INSTITUTE OF PHARMACEUTICAL
TECHNOLOGY & ALLIED HEALTH SICENCES
Affiliated to
Maulana Abul Kalam Azad University Of Technology, West Bengal
A Presentation on
RIA :Radio Immuno Assay
Presented by
RANITA PATRA
PRATIK SHEE
M. Pharmacy (1st year , 1st semester)
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Contents
Introduction
Principle
Requirements
Procedure
Application
Advantage & Disadvantage
Advancement
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Introduction
Immunoassay:
An immunoassay is a biochemical test used to identify the presenceor
amount of a particular molecule referred to as an "analyte”, in a
solution by combining it with an antibody or an antigen.
The principal of immunoassays is formation of an immune complex
involving the recognition and binding of an antibody to a specific
molecule among a mixture of molecules.
RIA (Radio Immunoassay):
RIA (Radio immunoassay) is a binding assay, in which the bounder is Figure : Basic components of an
an antibody, which uses radioactivity to measure the amount of bound Immunoassay.The analyte
specifically binds to the
and free antigen. antibody labeled with
RIA is a very sensitive, In-vitro assay technique. detectable label
It is generally used for measuring the concentration of an substance,
by using antibody (Example: Hormone level in blood).
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Principle of RIA:
RIA works on basic principle of biochemistry that competitive binding between antigens for
same antibody binding site.
The competition of an analyte with its radio isotopically 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. Ag: Ligand to be measured
Radioactivity of bound residue measured. Ag*:Radio labeled ligand
Ligand concentration is inversely related to radioactivity.
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Requirements of RIA:
.
Radiolabeled antigens: The antigens are generally labeled with gamma-ray
emitting isotopes such as I125 and beta-ray emitting isotopes such as Tritium.
They are also called hot antigens.
.
Specific Antibodies: They are required in smaller amounts than antigens.
Unlabeled antigens (sample antigens): They are also called cold antigens.
Microtitre plates: 96 wells microtiter plate.
Washing Buffer solutions: Wash buffer such as 1% Trifluoroacetic acid is
used.
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Procedure of RIA :
Specific antibodies of known concentration are fixed in
the microtiter well.
A known amount of hot antigens is then added two the
well.
Washed carefully to remove any unbound antigens.
At this point, the radioactivity of the wall be maximum.
Unlabeled antigens are then added to the wall.
The unlabeled antigens will bind to the antibodies and
there will be free labeled antigens in the well.
Again wash carefully to remove the free labeled
antigens.
Radioactivity of wells is then measure by gamma-
counter.
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Application Of RIA (Radioimmunoassay):
It was first used for the detection of peptide
hormones.
Detection of different viral antigens.
Detection of many hormones and drugs.
Detection of Hepatitis B surface antigens.
Detection of mycotoxins.
Detection of the early stage of cancer.
A radioimmunoassay (RIA) can be used to
diagnose a variety of diseases, including ;
1.Autoimmune disorders,Viral infections, Pancreatic
cancer ,Celiac disease, Cholestatic liver disease
and
Malaria .
2.RAST:It detects the allergen that causes an allergy.
3.Coated Tube RIA:It is used to detect drugs in
biological matrices.
Figure : An overview of Radioimmunoassay
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Advantages of RIA ( Radioimmunoassay):
• High specificity .
• High sensitivity .
•Quantitative & Precise.
• Can detect a very small amount (nanograms) of
antigen or antibodies.
Disadvantages of RIA ( Radioimmunoassay):
• Half life of I125 is 60 days only.
• Hazards of radioactivity.
• Lengthy procedure ,etc.
• Very high cost of equipments and reagents.
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Advancement Of RIA :
Radioimmunoassay (RIA) has seen many advancements, including;
Non-radioactive tracers
Non-radioactive tracers are expected to become more prevalent in future.
Solid-phase techniques
This techniques have been developed for both antigens and antibodies.
Cell cultures
Cell cultures are used to provide solid-phase reactants for viral antigens.
Sequence-specific assays
These assays are panels of assays that are specific for different sequences
of peptides.
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