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Basic steps of ELISA
Enzyme Linked Immunosorbent Assay
1. Antigen of interest is absorbed on to
plastic surface (‘sorbent’).
2. Antigen is recognised by specific
antibody (‘immuno’).
3. This antibody is recognised by second
antibody (‘immuno’) which has enzyme
attached (‘enzyme-linked’).
4. Substrate reacts with enzyme to produce
product, usually coloured.
Coloured product = measure
(assay) of antigen present
Types of ELISA
Antigen
Direct ELISA
Antibody-enzyme
Antigen
substrate
Antibody-enzyme
Antigen
Sandwich ELISA ("Indirect" ELISA)
substrate
Antibody-enzyme
Antibody
Antigen
Antibody
Antibody
Antigen
Antibody
Antibody
Antigen
Antibody
Competitive ELISA
1. Unlabeled antibody is incubated in the presence of its antigen (sample).
2. These bound antibody/antigen complexes are then added to an antigen-coated well.
3. The plate is washed, so unbound antibody is removed. (The more
antigen in
the sample, the more Ag-Ab complexes are formed and so there are
less unbound antibodies available to bind to the antigen in the well, hence
"competition".)
4. The secondary antibody, specific to the primary antibody, is added. This second
antibody is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent
signal.
6. The reaction is stopped to prevent eventual saturation of the signal.
• Microplates: Polystyrene – proteins absorb (bind)
by hydrophobic bonds to the polystyrene
• Secondary antibody: anti-human antibody linked
(conjugated) to horseradish peroxidase (HRP)
• Enzyme substrate: 3,3’,5,5’ – tetramethylbenzidine (TMB) – a colorless
solution that when oxidized by HRP turns yellow. A substrate is a compound
or substance that undergoes change.
– Substrates bind to active sites on the surface of enzymes and are
converted or changed. In ELISA the specific substrate used changes
color.
– Substrate Solution:
• chromogen A and
• chromogen B should be mixed together in equal volumes up to 15
minutes before use.
• STOP SOLUTION:
– (sulphuric acid solution (H2SO4).
Horseradish peroxidase
The
enzyme
horseradish
peroxidase (HRP), found in horseradish,
is used extensively in biochemistry
Applications
ability to amplify a weak signal and increase
detectability of a target molecule.
 Horseradish peroxidase is also commonly used in
techniques such as ELISA
 it is smaller, more stable and less expensive than other
popular alternatives such as alkaline phosphatase
Horseradish peroxidase cont….
Applications
• Horseradish peroxidase is a
– 44,173.9 dalton glycoprotein with
– 4 lysine residues for conjugation to a labelled molecule.
– It produces a coloured, fluorimetric or luminescent derivative
of the labeled molecule allowing it to be detected and
quantified.
– HRP is often used in conjugates
• conjugates (molecules that have been joined chemically) to
determine the presence of a molecular target. For example,
an antibody conjugated to HRP may be used to detect a
small amount of a specific protein in a ELISA.
Substrates
• Alone, the HRP enzyme, or conjugates there
presence must be made visible using
a substrate that when oxidized by HRP
using hydrogen peroxide as the oxidizing
agent, yields a characteristic change that is
detectable by spectrophotometric methods
Enzyme + substrate + H2O2
HRP
DIIMINE
chromogen A: contains hydrogen peroxide
chromogen B: containes tetramethylbenzidine (TMB)
• Numerous substrates for the horseradish
peroxidase enzyme have been described
commercialized to exploit the desirable features of
HRP.
• These substrates fall into several distinct categories.
• HRP catalyzes the conversion of chromogenic
substrates (e.g. TMB, ) into colored molecules, and
produces light.
3,3’,5,5’-Tetramethylbenzidine
 TMB can act as a hydrogen donor for the reduction
of hydrogen peroxide to water by peroxidase enzymes
such as horseradish peroxidase.
 The resulting diimine causes the solution to take on a
blue colour, and this colour change can be read on
a spectrophotometer at a wavelength of 650 nm.
 The reaction can be halted by addition of acid or another
stop reagent. Using sulfuric acid turns TMB yellow. The
colour may be read at 450 nm.
Material Safety
• TMB should be kept out of direct sunlight as it is
photosensitive.
• On that evidence, TMB has been used as a
replacement for carcinogenic compounds
such o-phenylene diamine.
Carcinogenic is a physical or chemical
agent that having the potential to cause
cancer.
mutagen is a physical or chemical agent that
changes the genetic material, usually DNA
Conjugated protein
simple proteins
 Many proteins contain only amino acids and no other chemical
groups, and they are called simple proteins.
conjugated protein
 A conjugated protein is a protein that functions in interaction
with other (non-polypeptide) chemical groups attached by
covalent bonding or weak interactions.
 The nonamino part of a conjugated protein is usually called its prosthetic
group.
 Conjugated proteins are classified on the basis of the chemical nature of
their prosthetic groups.
• Some examples of conjugated proteins
are lipoproteins, glycoproteins, phosphoproteins, hemoproteins, flavoprote
ins, metalloproteins, phytochromes, cytochromes and opsins.
• Spectrophotometry
– Spectrophotometry
involves
the
use
of
a
spectrophotometer.
– A spectrophotometer is a photometer (a device for
measuring light intensity) that can measure intensity as
a function of the light source wavelength.
– It is the quantitative measurement of the reflection or
transmission properties of a material as a function of
wavelength. It deals with visible light, near-ultraviolet,
and near-infrared,
Bovine serum albumin (also
known as BSA or "Fraction V")
• a serum albumin protein that has numerous biochemical
applications including ELISA and immunohistochemistry.
• It is also used as a nutrient in cell and microbial culture.
• In restriction digests,
– BSA is used to stabilize some enzymes during digestion of DNA
– prevent adhesion of the enzyme to reaction tubes and
other vessels.
– This protein does not affect other enzymes that do not
need it for stabilization.
Sample diluents
PBS: Phosphate buffered saline –
provides stable buffered environment to maintain
antibody structure
A PBS buffer containing
 0.02% Tween 20, protein as stabilizer and
0.05% Kathon CG as preservative.
Wash Buffer
• Wash Buffer(Tween 20)
1. detergent – removes non-specifically bound proteins
1.
to reduce background and blocks protein binding sites on the
polystyrene
If a smaller volume of Wash Buffer is desired,
add 1 volume of Wash Buffer (20X) to

19 volumes of distilled or deionized water.
Wash Buffer is stable for 1 month at 2-8°C. Mix well
before use.
How we will detect:
read absorbance at 450 nm
Absorbance 450 nm
1
A
B
W
E C
L
L D
E
F
G
H
2
3
4
5
6
7
8
9
10
11
NPTII
Std