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Principle of RIA
Theory
Requirements
Methods
Merits & De-Merits
Applications
Related Techniques
References
Radioimmunoassay (RIA) is a sensitive
method for measuring very small
amounts of a substance in the blood.
Radioactive versions of a substance, or
isotopes of the substance, are mixed
with antibodies and inserted in a
sample of the patient's blood.
The same non-radioactive substance in
the blood takes the place of the isotope
in the antibodies, thus leaving the
radioactive substance free.
Theamount of free isotope is then
measured to see how much of the original
substance was in the blood.
measurement of
insulin.
› 3H 14
C 125
I are used as radioactive
tags
› Antigens are tagged to 3 H 14 C 125
I
› Tagging should NOT affect
Antigenic specificity &
Antigenic activity!!!
Usually high specific activity
- radio-labeled (125-I) antigen is used -
prepared by
- iodination of the pure antigen on its
tyrosine residue(s)
- by chloramine-T or peroxidase methods
and then separating the radio-labeled
antigen from free-isotope by gel-filtration
or HPLC.
Some ligands are not Antigenic
› Hormones, Steroids, Drugs HAPTENS
› Eg: Gastrin, Morphine,
Various
separation methods are used
based on physical and biochemical
characters of the bound complexes.
Precipitation of the antibody
- Fractional Precipitation
› Centrifugation
› Filtration
Adsorption of the free Antigen
Use of solid phase Antibody
Gel Filtration
Adsorption Chromatography
Partition Chromatography
› Dialysis
Electrophoresis
Ultrafiltration
Precipitation of the antibody:
Used if mol. Size of B & F forms of Ag
differ considerably
Electrophoresis
Gel Filtration
Adsorption Chromatography
Fractional Precipitation
› Centrifugation
› Filtration
Partition Chromatography
› Dialysis are used…
Variousinstruments are used –
detection and measurement of
radioactivity.
› Scintillation counter
› Geiger-muller counter
› Gas counters etc..
Bothfree(F) and bound(B) fractions are
counted,
Non-Competitive Immunoassays
unlabelledanalyte in the test sample is
measured by its ability to compete with
labeled antigen for a limited number of
antibody binding sites.
The
amount of antigen in the test
sample is inversely related to the
amount of label measured in the
competitive format. As one increases,
Highest level of assay sensitivity and
specificity.
This
format is referred to as a
“sandwich” assay because the
analyte is bound (sandwiched)
between two highly specific antibody
reagents.
The reaction mixture typically includes an
excess of labeled antibody, so that all
drug/metabolite is bound. The amount of
antibody-antigen complex is then
measured to determine the amount of
drug present in the sample. The
measurement of labeled analyte, usually
antibody, is directly proportional to the
amount of antigen present in the sample.
Immunoassay methods that require
separation of bound Ab-Ag* complex -
heterogeneous immunoassays. Those
that do not require separation -
homogeneous immunoassays.
Epidemiology
› Hepatitis B
Clinical Immunology
› Antibodies for Inhalant Allergens
› Allergy Diagnosis
Oncology
› Carcinoembryonic Antigen
› Early Cancer Detection and Diagnosis
Enzyme Multiplied Immunoassay (EMIT )
Enzyme-linked immunosorbent
assay (ELISA)
Enzyme activity/absorbance is
directly proportional to drug
- competitive, heterogeneous EIA