Transcript lecture-4-radioimmunassay

Radioimmunoassay
• Radioimmunoassay (RIA) involves the
separation of a protein (from a mixture)
using the specificity of antibody - antigen
binding and quantitation using
radioactivity.
• The technique of radioimmunoassay
has revolutionized research and
clinical practice in many areas, e.g.,
– blood banking
– diagnosis of allergies
– endocrinology
• The technique was introduced in 1960 by
Berson and Yalow as an assay for the
concentration of insulin in plasma.
• It represented the first time that hormone
levels in the blood could be detected by an
in vitro assay.
Principle
• Based on competition between unlabelled
antigen and finite amount of corresponding
labelled antigen for a limited number of
antibody binding sites in a fixed amount of
antiserum.
• At equilibrium in the presence of an
antigen excess there will be both free
antigen antigen bound to antibody.
• Under standard conditions the amount of
labelled antigen bound to the antibody will
decrease as the amount of unlabelled
antigen in the sample increases.
• 4Ag⃰ + 4 Ab → 4Ag⃰.Ab
• 4 Ag + 4 Ag⃰ + 4 Ab → 2Ag⃰Ab + 2 AgAb +
2Ag⃰ + 2Ab
• 12 Ag + 4 Ag⃰ + 4 Ab → Ag⃰ Ab + 3AgAb +
3Ag⃰ + 9 Ag
• Principle: Uses an immune reaction
• [Antigen – Antibody reaction] to estimate a ligand
Ag + Ag* + Ab  AgAb + Ag*Ab + Ag + Ab*
– Unbound Ag* and Ag washed out
– Radioactivity of bound residue measured
– Ligand conc is inversely related to radioactivity
[Ag : ligand to be measured ; Ag* radiolabelled ligand]
• At increasing concentrations of unlabeled
antigen, an increasing amount of
radioactive antigen is displaced from the
antibody molecules.
• The antibody-bound antigen is separated
from the free antigen in the supernatant
fluid, and the radioactivity of each is
measured.
Gamma Counter
• From these data, a
standard binding
curve, like the one
shown in red, can be
drawn.
• The samples to be
assayed (the
unknowns) are run in
parallel.
• After determining the
ratio of bound to free
antigen in each
unknown, the antigen
concentrations can be
read directly from the
standard curve.
• The main drawbacks to radioimmunoassay are
the expense and hazards if preparing and
handling the radioactive antigen.
• Both 125I or 131I emit gamma radiation that
requires special counting equipment;
• The body concentrates iodine atoms —
radioactive or not — in the thyroid gland where
they are incorporated in thyroxine (T4).
• Despite these drawbacks, RIA has become a
major tool in the clinical laboratory where it is
used to assay
• plasma levels of:
– most of our hormones;
– digitoxin or digoxin in patients receiving these drugs;
– certain abused drugs
• for the presence of hepatitis B surface antigen
(HBsAg) in donated blood;
• anti-DNA antibodies in systemic lupus
erythematosus (SLE).