Questions on RIA and ELISA
1. RIA
2. ELISA
3. Radioimmunoassay
4. Principles and applications of RIA
5. Principles and applications of ELISA
6. Applications of ELISA
7. Applications of RIA
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Radioimmunoassay (RIA)
[Note: Test inside the box is for deeper understanding of the subject and not for
examination]
This is a clinical biochemical assay for determination of concentration of antigenic
substances in body fluids by antigen-antibody reaction using an appropriate antibody
and radio-labeled antigen.
Radioimmunoassay, thus, combines the advantages of specificity of the antigenantibody interaction and sensitivity of measurement of radio-labeled compounds.
Principle
Radioimmunoassay is based on competitive binding of antigen (analyte, unlabeled
antigen present in the serum sample to be assayed) and radio-labeled antigen to
antibody.
125
I and 131I are the frequently used radioisotopes in radioimmunoassays for labeling.
The extent binding of the labeled antigen to the antibody is then inversely
proportional to the concentration of analyte antigen present in the serum sample.
The amount of labeled antigen present in the antibody-bound antigen is determined by
measuring the radioactivity.
The radioactivity measured will be also inversely proportional to the
concentration of antigen present in the sample.
Applications
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RIA is used in the estimation of substances that exhibit antigenic property
either as such or by chemical modification.
Substances that are estimated by RIA are hormones, tumor markers (peptides,
proteins, steroids, etc), vitamins, drugs, etc.
Thus, radioimmunoassay has applications in the diagnosis of hormonal
disorders, cancers and therapeutic monitoring of drugs.
It is also useful in biomedical research.
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Principle of RIA
Radio-labeled antigen is in excess but of fixed quantity, antibody is in limited
quantity and the quantity of the analyte antigen (unlabeled present in the
sample to be assayed) is unknown.
Antigen (analyte, unlabeled, unknown quantity in the sample)
Antigen (Unlabeled)
Reaction
Antibody
Antigen-Antibody
(Limited quantity)
(Unlabeled)
Antigen*-Antibody
Antigen*
(Labeled)
(Labeled, fixed quantity, in excess)
Antigen* (Labeled)
After reaching equilibrium in the antigen-antibody reaction,
antibody-bound antigen is separated from free antigen (labeled
and unlabeled).
Radioactivity 
A standard curve is drawn by using different known
concentrations of unlabeled antigen and same quantities of
antibody and labeled antigen. This can be used, by comparison,
to derive antigen concentrations in samples.
Y
0
X
Concentration 
(of unlabeled antigen standards)
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Example – 1
Antibody
Reaction
After equilibrium
Analyte, unlabeled antigen
Labeled antigen
Example – 2
Reaction
After equilibrium
Enzyme-Linked Immunosorbant Assay (ELISA)
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ELISA is a non-isotopic immunoassay for both detection and quantitation of
antigens or antibodies in body fluids for clinical diagnosis.
An enzyme is used as a label in ELISA in place of radioactive isotope
employed in RIA.
ELISA is as specific as RIA (though less sensitive). In addition, there is no
risk of radiation hazards in ELISA (as is the case with RIA.
Principle
ELISA is used for both detection and quantitation of antigens or antibodies in
body fluids for clinical diagnosis.
Assay for Antigens
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Antibody to the analyte antigen to be assayed is fixed to a solid surface
(microtiter well).
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Then the analyte antigen (to be assayed present in serum) binds to the
antibody.
And then enzyme-labeled second antibody binds to the antigen.
Thus the analyte antigen is sandwiched between two antibodies.
The amount of enzyme-linked antibody getting fixed to the well will be
directly proportional to the amount of antigen present in the serum.
The enzyme selected to label is such that it can catalyze formation of a colored
substance (chromogen) from a substrate.
Intensity of color developed, measured colorimetrically, will be directly
proportional to quantity of the enzyme-labeled antibody fixed to the well,
which in turn is proportionate to quantity of analyte antigen present in
the serum.
First antibody
Enzyme
Microtiter Well
Immobile Solid Surface
antibody
Substrate
Antigen (Serum)
Enzyme-labeled second
Color Product
Enzyme
Enzyme-labeled Second antibody
Antigen (Serum)
First antibody
Microtiter Well
Immobile Solid Surface
Assay for Antibody
In this the analyte is an antibody and it is sandwiched between an antigen and
enzyme-labeled antibody to the antibody to be assayed.
Applications
In clinical practice ELISA is used for quantitation and detection of proteins including
antigens, antibodies, hormones and tumor markers.
Examples:
Antibodies – e.g. antibodies against HIV for test for AIDS
Antigens – e.g. detection of HIV antigen
Hormones – e.g. human chorionic gonadotropin (hCG) for pregnancy test.
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