Download 7th elisa

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
Capture and Detection Antibodies
HCV ELISA
• Classical, parentally transmitted non-A, non-B
hepatitis has been shown to be due to a small
RNA virus, HCV. Non-A, non-B hepatitis
represents greater than 90% of transfusionassociated hepatitis cases in the world, and up
to 10% of transfusions have been estimated to
result in non-A, non-B hepatitis.
• This HCV ELISA kit has succeeded in detecting
HCV antibodies by using a highly antigenic
polyprotein derived from genes encoding
essential epitopes sequences of HCV strains
prevalent in several regions of the world. It may
provide a more comprehensive and effective
diagnostic assay to HCV antibodies
Test Principle
• The test principle is based on indirect enzyme immunoassay.
• Micro titer wells are coated with certain amount of HCV recombinant
antigens including NS3,NS4,NS5 and CORE antigens.
• Then serum samples are allowed to react with solid phase antigens.
• If HCV-specific antibodies (IgG and IgM) are presented in the serum
they will bind to HCV antigens through their individual Fab section.
• After incubation, the wells are washed to remove unbound
antibodies and anti-human antibodies (IgG/IgM) conjugated with HRP
is added into the wells following another incubation and wash step.
• A solution of TMB is added and incubated for 15 minutes, resulting in
the development of a blue color. The color development is stopped
with the addition of stop solution, and the color is changed to yellow
and measured spectrophotometrically at 450 nm.
• The concentration of specific anti HCV is directly proportional to the
color intensity of the test sample.
Horseradish peroxidase
• The enzyme horseradish peroxidase (HRP),
found in horseradish, is used extensively
in biochemistry applications primarily for its
ability to amplify a weak signal and increase
detectability of a target molecule.
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 (molecules that
have been joined genetically or 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 western blot.
• Here, the antibody provides the specificity to
locate the protein of interest and the HRP
enzyme, in the presence of a substrate,
produces a detectable signal.
• Horseradish peroxidase is also commonly used
in techniques such
as ELISA and Immunohistochemistry.
• Horseradish peroxidase is ideal in many respects for
these applications
• because it is smaller, more stable and less
expensive than other popular alternatives such
as alkaline phosphatase.
• It allows generation of strong signals in a relatively
short time span.
Substrates
• Alone, the HRP enzyme, or conjugates there
of, is of little value; its 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
• 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, DAB, ABTS) into colored
molecules, and produces light when acting
on chemiluminescent substrates (e.g. SuperSignal,
ECL).
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.
• It is not known if TMB is carcinogenic and the
evidence is contradictory:
• TMB is not mutagenic
• On that evidence, it has been used as a
replacement for carcinogenic compounds such
as benzidine and o-phenylenediamine.
•
Conjugated protein
conjugated protein is a protein that functions in
interaction with other chemical groups attached
by covalent bonds or by weak interactions.
• Many proteins contain only amino acids and no other
chemical groups, and they are called simple proteins.
However, other kind of proteins yield, on hydrolysis, some
other chemical component in addition to amino acids and
they are called conjugated proteins. 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, hemopr
oteins, flavoproteins, metalloproteins, phytochromes, cyto
chromes and opsins.
• In chemistry, spectrophotometry is the quantitative
measurement of the reflection or transmission
properties of a material as a function of wavelength. It
is more specific than the general term electromagnetic
spectroscopy in that spectrophotometry deals
with visible light, near-ultraviolet, and near-infrared,
• 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.
Important
features
of
spectrophotometers are spectral bandwidth and linear
range of absorption measurement.
• Spectrophotometers are most commonly used for the
measurement of transmittance or reflectance of a
solution or transparent material, like polished glass.
However they can also be designed to measure
the diffusivity on any of the listed light ranges that
usually cover around 250nm - 2500nm using different
controls and calibrations.
• Within these ranges of light, calibrations are needed
on the machine using standards that vary in type
depending on the wavelength of the photometric
determination
• The use of spectrophotometers spans various
scientific
fields,
such
as physics, chemistry, biochemistry, and molecular
biology.
• Bovine serum albumin (also known as BSA or "Fraction V")
is a serum albumin protein that has numerous biochemical
applications
including
ELISAs(Enzyme-Linked
Immunosorbent
Assay),
immunoblots,
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 and to
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. BSA is
also commonly used to determine the quantity of other
proteins, by comparing an unknown quantity of protein to
known amounts of BSA. BSA is used because of its stability,
its lack of effect in many biochemical reactions, and its low
cost since large quantities of it can be readily purified from
bovine blood, a byproduct of the cattle industry.
• The nickname "Fraction V" refers to albumin
being the fifth fraction of the original Edwin Cohn
purification methodology that made use of
differential solubility characteristics of plasma
proteins. By manipulating solvent concentrations,
pH, salt levels, and temperature, Cohn was able
to pull out successive "fractions" of blood plasma.
The process was first commercialized with human
albumin for medical use and later adopted for
production of BSA.
• Sample diluents: A PBS buffer containing
0.02% Tween 20, protein as stabilizer and
0.05% Kathon CG as preservative. 1. PBS:
Phosphate buffered saline – provides stable
buffered environment to maintain antibody
structure
• Wash Buffer:( Tween 20) Nonionic detergent –
removes non-specifically bound proteins to
reduce background and blocks protein binding
sites on the polystyrene
• Add 60 mL of Wash Buffer (20X) and dilute to a
final volume of 1200 mL with distilled or
deionized water. Mix thoroughly. 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.
• 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. Refer
to the table provided for correct amounts of Substrate Solution to
prepare.
• chromogen A: contains hydrogen peroxide
• chromogen B: containes tetramethylbenzidine (TMB)
• STOP SOLUTION: (2N sulphuric acid solution (H2SO4).