Transcript Document

A SEMINAR ON
IMMUNO-DIAGNOSTIC
TECHNIQUES
PRECIPITATION REACTIONS
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In Fluids
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In Gels
RADIAL IMMUNODIFFUSION
DOUBLE IMMUNODIFFUSION
AGGLUTINATION
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The interaction between antibody and a
particulate antigen results in visible clumping
called agglutination. Ab that produce such
reaction are called agglutinins.
RADIOIMMUNOASSAY
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First developed by S. A. Berson and Rosalyn Yalow in
1960 to determine levels of insulin-anti-insulin complexes
in diabetics
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RIA has been widely used to screen for the
presence of the hepatitis B virus.
It can also be used to detect the levels of most
of our hormones, digitoxin or digoxin in
patients receiving these drugs.
It can also be used to detect the concentration
of certain abused drugs.
RIA can also be used for the detection of
specific secretory protein tuberculin derived
from Mycobacterium tuberculosis.
ELISA
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ELISA PLATE
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ELISA are created by coating
the antigen or antibody on a
suitable plastic. To complete
the reaction, an enzymatic
detection method with a
color-forming substrate is
required.
ELISA can be either
competitive or
noncompetitive.
ELISA is a standard tool for
quantifying the antibody or
antigen in a serum.
INDIRECT ELISA
Primary antibody is added to an
antigen-coated microtiter well.
After any free Ab is washed
away, the presence of antibody
bound to the antigen is
detected by adding an enzyme
conjugated
secondary
antiisotype antibody (Ab2)
which binds to the primary
antibody. Any free antibody is
then washed away and a
substrate for the enzyme is
added and the colored reaction
product is measured by
spectrophotometric
plate
reader, which can measure the
absorbance of all the wells.
SANDWICH ELISA
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In this technique, the
antibody is immobilized on
a microtiter well.
Sample containing antigen
is added and allowed to
react with the immobilized
antibody.
Second enzyme linked
antibody specific for a
different epitope on the
antigen is added.
Substrate
added
and
colored reaction product is
measured.
COMPETITIVE ELISA
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The ELISA, or the enzyme immunoassay (EIA), was the
first screening test widely used for HIV because of its
high sensitivity.
In an ELISA, a person's serum is diluted 400-fold and
applied to a plate to which HIV antigens are attached. If
antibodies to HIV are present in the serum, they may
bind to these HIV antigens. The plate is then washed to
remove all other components of the serum.
Pregnancy tests also use a sandwitch ELISA method.
An enzyme immunoassay (EIA) for detection of serum
antibodies in patients with typhoid fever was developed
using Salmonella typhi outer membrane protein (OMP)
preparations as antigen.
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