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elisa is short for enzyme-linked
immunosorbent sa with this
plate-based technique we can detect and
quantify
soluble substances such as proteins
during an elisa the antigen which is the
target molecule
is immobilized and specifically detected
by antibodies
which are enzyme-coupled when providing
a colorless substrate the enzyme
produces
a measurable colorful product an
antibody is attached to the bottom of
the well
this antibody is specifically designed
for binding a specific protein of
interest
another soluble antibody is provided
the antibody is also specifically
covering protein
x this antibody is conjugated to an
enzyme when we now add a substrate
the enzyme can convert it to a colorful
product
which can be measured later there are
many variants of an elisa
in the direct elisa the antigen
is immobilized on the bottom of the
plate and
the enzyme-linked antibody directly
binds to the antigen
in the indirect elisa a primary antibody
binds our antigen
and then a secondary antibody binds the
primary antibody
the secondary antibody is enzyme linked
the most common type is the sandwich
elisa
a primary antibody is immobilized and
captures the antigen of a solution and
another primary antibody
specifically binds the same antigen but
all three elisa's
have one thing in common the last
antibody binding
is always conjugated to the enzyme which
confirms binding here we provide an
example of an elisa
for this sandwich elisa we have two
wells
both coated with primary antibody
specific for protein x on the left
we provide a sample containing our
protein
on the right we have the control without
the target protein
our antigen in orange will be
immobilized
a washing step will discard everything
else
now we provide the enzyme-linked
antibody
it will bind our antigen in the left
well and
remains bound after washing whereas the
antibody is discarded in the control
in the last step a colorless substrate
is given
since the enzyme in most cases
horseradish peroxidase
is exclusively present in the left well
it produces a measurable colorful
product here
this can be investigated and also
quantified
using spectophotometry the control
remains colorless since substrate is not
converted here if you want to have a
closer look into the direct or the
indirect elisa
you may click one of these videos here
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