Elisa Flashcards
is a widely used biochemical assay to detect in a
sample the presence of and quantity of proteins, such as
hormones and antibodies and bacteria or viruses.
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
uses the coupling of antigens and antibodies and relies on the
specificity and affinity of antibodies for antigens. Specificity is
the ability to discriminate among diverse proteins. Affinity is the
ability to tightly bind to molecules.
ELISA
One can determine how much antibody is present by starting
with an antigen, or one can determine how much antigen or
hormone is present by starting with an antibody.
ELISA
are large glycoprotein molecules produced by Blymphocytes during the humoral
immune response to
antigens introduced into the body.
Antibodies
include
B-lymphocytes
(B-cells)
and
Tlymphocytes (T-cells) which are white blood cells form from
the hematopoietic (blood) stem cells in the bone marrow. .
Lymphocytes
(1)
Antibodies bind to antigens following the lock-and-key
model. Antigens bind to the receptor sites.
• (2)_____can also bind to antibodies following
the same principle. These can either bind to the receptor site
or the tail part.
(1)Antibody -Antigen interaction
(2) secondary antibodies
Antigen/antibody
of
interest
is
adsorbed on a plastic surface
(sorbent)
Antigen is recognized and binds to a
specific antibody
Immuno
The antibody is recognized by the
second antibody which has an
enzyme attached
Enzyme-linked
Substrate react with the enzyme to
produce a product, usually _____
Colored
has been used to
detect hepatitis B, rabies, and HIV
through antibodies in the blood serum,
just to name a few diseases, or to
measure the amount of various other
proteins in the blood serum, such as
hormones, toxins, and allergens.
ELISA method
detect antibodies in the sample
• a) Binding Known Antigen
• b) Blocking
• c) Washing
• d) Adding Test Sample Primary Antibody
• e) Washing
• f) Adding Enzyme-linked Secondary Antibody
• g) Washing
• h) Adding Substrate
• i)
Reading
Results
The indirect ELISA
method begins with a sample of known
antigen being bound to the wells of a microtiter
plate.
Binding Known Antigen
he other unoccupied sites in
each well are then bound by a concentrated
solution of non-interacting protein, like casein
or bovine serum albumin, to block or prevent
other proteins in the test sample from adhering.
Blocking
Rinse to remove any unbound
antigen and non-interacting protein.
Washing
The
test sample of serum containing the primary
antibodies is added to each well. Antibodies
could be HIV, rabies, or hepatitis B antibodies,
for example.
Adding Test Sample Primary Antibody
Rinse to remove any antibodies
that did not bind to the known antigen.
Washing
An enzyme-linked
secondary antibody is
added next to bind to the test sample
antibodies. The enzyme on the secondary
antibodies are proteins, such as horse radish
peroxidase or alkaline phosphatase.
Adding Enzyme-linked Secondary Antibody
Rinse to remove any secondary
antibodies that did not bind to the primary
antibody.
Washing
A substrate is then
applied which is converted by the enzyme to
give a color or fluorescence or electrochemical
signal. In the presence of horse radish
peroxidase, TMB turns blue.
Adding substrate
By
using
a
spectrophotometer,
spectrofluorometer,
or electrochemical device, the results can be
read and recorded. The amount of color
produced is proportional to the amount of
primary antibody bound to the antigen proteins
on the bottom of the wells.
Reading results
plays the role of
a chromogenic substrate, and is also one of the most
sensitive substrates for HRP. When a TMB solution is added to
HRP, HRP will reduce hydrogen peroxide and oxidize TMB,
turning it from colorless to blue-green.
TMB (3,3’,5,5’-Tetramethylbenzidine)
are commonly
used to read ELISA results.
Spectrophotometers (microplate reader)
has the advantage of signal
amplification
Indirect ELISA
detect antigens in the sample
• This is the most commonly used format.
• This format requires two antibodies specific for different epitopes
of the antigen. These two antibodies are normally referred to as
matched antibody pairs.
• Highly specific which minimizes false positives
SANDWICH ELISA
it is common for ELISAs to
detect antigens at the picogram level in a very specific
manner due to the use of antibodies.
ELISA :High sensitivity and specificity
commercial ELISA kits are normally
available in a 96-well plate format. But the assay can be easily
adapted to 384-well plates.
ELISA: High throughput
protocols are easy to follow and involve little
hands-on time.
ELISA: Easy to perform
it can determine the concentration of antigen
in a sample. • Possibility to test various sample types: serum,
plasma,
ELISA: Quantitative
detection is based on enzyme/substrate
reactions and therefore readout must be obtained in a short
time span.
ELISA: temporary readouts
information is limited to the
amount or presence of the antigen in the sample.
ELISA: Limited antigen information
are techniques for transferring
DNA, RNA and proteins onto a carrier so
they can be separated, and often
follows the use of a gel electrophoresis.
Blots/blotting
• Southern blot – (1)
• Northern blot – (2)
• Western blot – (3)
• Eastern blot – (4)
(1)DNA
(2) RNA
(3)Proteins (immunoblot)
(4) post-translational proteins
is an analytical method
that involves the immobilization of
proteins
on
membranes
before
detection using antibodies
Protein blotting
is
a
prerequisite for Western blotting
SDS
PAGE
technique
(1)____is an
electrophoresis
method that allows
protein separation by
mass.
SDS-PAGE
(1) Polyacrylamide Gel
Electrophoresis
is
a
negatively
charged
detergent
used
to
denature
and linearize proteins
Sodium
dodecylsulfate (SDS)
Application of electric
field
causes
the
migration
and
separation of proteins
SDS-PAGE
is used
to form a gel that
provides a matrix of
pores through which
molecules migrate at
different rates
Polyacrylamide
• Visualize
the
band
under UV
• Visualize by staining
• Coomassie Blue
• Silver stain
VISUALIZATION OF PROTEIN BANDS
• Coomassie Blue-(1)
• Silver stain-(2)
(1)most common
(2)most sensitive test
In
order
to
make
proteins accessible to
antibody
detection,
they
are transferred
onto
a
membrane
made of nitrocellulose
or
polyvinylidene
difluoride (PVDF)
WESTERN BLOT - TRANSFERRING
WESTERN BLOT - TRANSFERRING
• Diffusion transfer
• Electro transfer
Western blotting transferring
SDS-PAGE ->transfer to a membrane ->blocking and reaction with antibodies -> detection of bands
