Antigen antibody
reactions
Antigen antibody
reactions
Ag-Ab reaction occurs in three stages:
Primary Stage
Formation of Ag-Ab complex combined by weaker
intermolecular forces
Secondary stage
leads precipitation
agglutination
lysis of cells etc.
Tertiary stage (reaction):
Leads to tissue damage
Destruction of Ag or its
Neutralization
CLASSIFICATI
ON
Primary binding - directly measure the
binding of antigen to antibody e.g. RIA, IF, ELISA.
test
Secondary binding test - measure the results of
antigen – antibody interaction in vitro, e.g.
precipitation, complement fixation.
In vivo test - measures the actual protective effect of
antibodies in a host, e.g. passive cutaneous
anaphylaxis.
RADIO IMMUNO
ASSAY
(RIA)
RADIO IMMUNO ASSAY
RIA was developed by Barson and Yalow in 1960
Thetest use radioactive isotopes for detecting antigen or
antibody. The commonly used isotopes are H3, C14, or I125
RIA is one of the most sensitive immunological technique
Uses of Radioimmunoassay
The test can be used to determine very small quantities of antigens and antibodies in the
serum (anti DNA Abs in SLE).
The test is used for quantitation of hormones, drugs, HBsAg, and other viral antigens.
Analyze nanomolar and picomolar concentrations of hormones in biological fluids.
Limitations of Radioimmunoassay – expensive (gamma counters), hazards of radioactivity, limited
shelf-life of labeled reagents, availability of radioisotopes and environmental concerns with safe
disposal of radioactive material
Principle of
Radioimmunoassay
It involves a combination of three principles.
An immune reaction i.e. antigen, antibody binding.
A competitive binding or competitive displacement reaction. (It gives
specificity)
Measurement of radio emission. (It gives sensitivity)
The classical RIA methods are based on the principle of
competitive binding:
an unlabeled antigen competes with a radiolabeled antigen for binding
to an antibody with the appropriate specificity
the amount of free (not bound to antibody) radiolabeled antigen is directly proportional
to the quantity of unlabeled antigen in the mixture.
Technique of
Radioimmunoassay
A mixture is prepared of
radioactive antigen (“hot")
antibodies ("First" antibody) against that antigen.
Known amounts of unlabeled ("cold") antigen are added to samples of
the mixture. These compete for the binding sites of the antibodies.
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.
From these data, a standard binding curve can be drawn
The samples to be assayed (the unknowns) are run in parallel.
After determining the ratio of bound to free antigen ("cpm Bound/cpm
Free") in each unknown, the antigen concentrations can be read
directly from the standard curve
Technique of
Radioimmunoassay
Separating Bound from Free Antigen
Precipitate the antigen-antibody complexes adding a
"second"
by antibody directed against the first. For
example,
rabbit IgG and anti rabbit-IgG
The antigen-specific antibodies can be coupled to the inner walls
of a test tube and after incubation:
the contents ("free") are removed;
the tube is washed ("bound"), and
the radioactive of both is measured.
The antigen-specific antibodies can be coupled to
particles,
like Sephadex. Centrifugation of the reaction mixture separates
the bound counts (in the pellet) from
the free counts in the supernatant fluid.
Technique of
RIA
Standard curve
Separating antigens by precipitation
Competitive RIA for Ag
detection
Determine amount of Ab
needed to bind to a known
Prior to Test
amount of labeled Ag
Use predetermined
amounts of labeled Ag and +
Ab and add a sample
containing unlabeled Ag as Labeled Ag
a competitor Test
Determine amount of
labeled Ag bound to Ab
+ +
Concentration of Ag in
sample is determined +
from a standard curve
using known amounts of Labeled Patient’s
unlabeled Ag Ag sample
Non- Competitive RIA for Ab
detection
Immobilize Ag
Labeled
Incubate with patient’s Anti-Ig
serum sample Ab in
Patient’
Add labeled anti-Ig s
sample
Amount of labeled Ab Immobilized Ag
bound is proportional to
amount of Ab in the Solid
sample Phase
Non- Competitive RIA for Ag
detection
Immobilize Ab Labeled
Ab
Incubate with patient’s
Ag in
sample containing antigen Patient’
Add labeled antibody s
Ag
sample
Amount of labeled Ab bound Immobilized
is proportional to the
amount of Ag in the sample Solid
Phase
