IMMUNOBLOTTING

TECHNIQUES-

 ELISA

 Western blotting,

 Southern blotting
IMMUNOASSAY

 A technique that utilizes the

specificity of binding between an
antigen and antibody in order to
identify and quantify the specific
antigen (or antibody) in a sample.
 For the purpose of quantification,
one of the components (Ag or Ab)
needs to be labeled
 The label produces a signal which
can be measured
 Initial assays used radioactive label
– RIA
 Currently non-isotopic labels being
used
APPLICATIONS OF IMMUNOASSAYS
 Infections: * Bacterial- M.tuberculosis, H.pylori * Viral-
HIV, Hepatitis B Virus
 Analytes: * Hormones- TSH, insulin, beta hCG
 * Vitamins- Vitamin D3 * Transport proteins- Transferrin
 Autoantibodies: * SLE, Rheumatoid arthritis
 Cancer biomarkers: * PSA, CA 125, CEA, CA 19.9
 Drug monitoring: * Therapeutic- Digoxin, Phenytoin
* Illicit - Heroin, performance enhancers in sports
ENZYME LINKED IMMUNOSORBENT
ASSAY (ELISA)
 ELISA stands for "enzyme-linked
immunosorbent assay."
 This is a rapid immunochemical test
that involves an enzyme (a protein that
catalyzes a biochemical reaction). It
also involves an antibody or antigen
(immunologic molecules).
 ELISA tests are utilized to detect
substances that have antigenic
properties, primarily proteins (as
opposed to small molecules and ions
such as glucose and potassium). Some
of these include hormones, bacterial
antigens and antibodies
 Based on specificity of antigen - antibody interaction
 Antigen of interest is adsorbed on to plastic surface
(‘sorbent’)
 Antigen is recognized by specific antibody (‘immuno’)
 This antibody is recognized by second antibody
(‘immuno’) which has enzyme attached (‘enzyme-
linked’)
 Substrate reacts with enzyme to produce product,
usually colored.
DIRECT ELISA

 Antigen to be detected is coated

directly
 Non specific sites are blocked

 Antibody labelled with enzyme is

added
 Wash to remove unbound antibody

 Substrate for the enzyme is added

 Colour development is proportional to

the amount of antigen present
 E.g. Detecting urinary beta hCG
SANDWICH ELISA
 Used to detect antibody
 Antigen is coated in a well

 Sample containing specific antibody to

be measured, is added
 Wash given to remove unbound antibody

 Secondary antibody (labelled with

enzyme) against the primary antibody is
added
 Substrate for the enzyme is added

 Colour development is proportional to

the amount of antibody present
 E.g. Detection of anti HBV antibody in
serum
 SANDWICH ELISA
 Used to detect antigen
 Capture antibody is coated in the well

 Sample containing Ag to be detected is

added
 Wash given to remove unbound antigen

 Detection antibody (labelled with

enzyme), against a different epitope of
antigen, is added
 Wash given to remove unbound antibody

 Substrate for the enzyme is added

 Colour development is proportional to the

amount of antigen present
 Eg. Estimation of serum insulin
COMPETITIVE ELISA
 Antibody is first incubated in solution with a
sample containing antigen
 This Ag-Ab mixture is then added to an Ag
coated well.
 More the Ag present in the sample, lesser is
amount of antibody available to bind to the Ag-
coated well
 Wash given to remove unbound Ag-Ab
complex
 Secondary Ab (labeled with enzyme) against
primary Ab is added
 Substrate for the enzyme is added
 Colour development is inversely proportional
to the amount of antigen present
 Used to detect weak immunogens e.g. hapten
APPLICATIONS OF ELISA
SUMMARY OF ELISA
 BLOTTING TECHNIQUES
 Visualization of specific DNA , RNA & protein among many
thousands of contaminating molecules requires the convergence of
number of techniques which are collectively termed BLOT transfer
 Involves electrophoretic separation of a nucleic acid or protein
mixture on an gel
 Separation followed by transfer of nucleic acid or protein bands onto
a support matrix (nitrocellulose/ nylon membrane) by electrophoresis
 Transferred nucleic acid or protein bands detected by treating the
membrane with radiolabeled or enzyme-linked probes or monoclonal
Abs specific for the target of interest
 Finally the target-probe complexes that form on the band containing
the nucleic acid or protein is recognized or visualized by: -
autoradiography - addition of a chromogenic substrate - addition of a
luxogenic substrate (chemiluminescence)
 SOUTHERN BLOT
 Transfer of DNA bands from an agarose gel to a membrane makes use
of the technique perfected in 1975 by Professor E.M. Southern and
referred to as Southern transfer
 The membrane is placed on the gel, and buffer allowed to soak
through, carrying the DNA from the gel to the membrane where the
DNA is bound.
 The same method can also be used for the transfer of RNA molecules
(“Northern” transfer) or proteins (“Western” transfer)
 If the labeled probe is now applied, hybridization occurs and
autoradiography (or the equivalent detection system for a non-
radioactive probe) reveals which restriction fragment contains the
cloned or target gene.
 IMMUNOBLOTTING

 Viral antigens are detected with a polyclonal or a MAb onto

nitrocellulose paper.
 After incubation, the protein bands (immune complexes) are
visualized with peroxidase-conjugated protein and a colour
reagent.
 A colour develops in the bands where antibody binds to the
antigen.
 Immunoblotting assay mixture of this two technique.
 WESTERN BLOTTING

 Western blotting is based on the principles of immunochromatography

where proteins were separated into poly acrylamide gel according to the
isoelectric point and molecular weight.
 A technique for detecting specific proteins separated by electrophoresis by
use of labeled antibodies.
 Immunoblotting is performed chiefly in diagnostic laboratories to identify
the desirable protein antigens in complex mixtures.
 An improved immunoblot method Zestern analysis, is able to address this
issue without the electrophoresis step, thus significantly improving the
efficiency of protein analysis.
 Other related techniques include dot blot analysis, zestern analysis,
immunohistochemistry where antibodies are used to detect proteins in
tissues and cells by immunostaining and enzyme-linked immunosorbent
assay (ELISA)
WESTERN BLOTTING

A technique to detect the presence of a

specific protein in a complex mixture
extracted from cells
Three key elements accomplish this
task: 1.separation of protein mixtures
by size using gel electrophoresis .
2. efficient transfer of separated
proteins to a solid support .
3.specific detection of a target protein
by appropriately matched antibodies.
Western Blot
Lane1: Positive Control
Lane 2: Negative Control
Sample A: Negative
Sample B: Indeterminate
Sample C: Positive
CONTENTS

 Cell / tissue lysis

 Protein quantitation
 Electrophoresis
 Transfer to a support matrix
 Detection
 Data analysis
 Applications
TISSUE PREPARATION

 Samples may be taken from whole tissue, from cell

culture, bacteria, virus or environmental samples.
 In most cases, samples are solid tissues.
 First broken down mechanically using a blender (for
larger sample volumes), using a homogenizer (smaller
volumes), or by sonication.
 Cells may also be broken open by one of the above
mechanical methods.
 A combination of biochemical and mechanical techniques,
including various types of filtration and centrifugation.
 To encourage lysis of cells and to solubilize proteins, may
be employed : detergents, salts, and buffers
 To prevent the digestion of the sample by its own enzymes
-Anti Protease and phosphatase
 To avoid protein denaturing-Tissue preparation is often
done at cold temperatures
GEL ELECTROPHORESIS

 The proteins of the sample are

separated using gel
electrophoresis.
 Separation of proteins may be by
isoelectric point molecular
weight, electric charge, or a
combination of these factors.
 Commercially SDS-PAGE gel
electrophoresis for protiens.
PAGE
 If the proteins are denatured and put into an electric field
(only), they will all move towards the positive pole at the
same rate, with no separation by size.
 However, if the proteins are put into an environment that
will allow different sized proteins to move at different
rates.
 The environment is polyacrylamide.

-The entire process is called polyacrylamide gel (PAGE)

electrophoresis.
Small molecules move through the polyacrylamide forest
faster than big molecules, big molecules stays near the well.
POLYACRYLAMIDE GEL
Polymerized gel:
 Resolving gels made in 6%,
10%, 12%, 18%.
 Stacking Gel up to 4 - 5% was
added to gel and then the wells
are created.
 The percentage chosen depends
on the size of the protein that
one wishes to identify or probe
in the sample.
 The smaller it is the bigger the
percentage.
SDS-PAGE (POLYACRYLAMIDE GEL ELECTROPHORESIS)

 SDS-PAGE, sodium dodecyl sulfate

polyacrylamide gel electrophoresis, is a technique
widely used in biochemistry, forensics, genetics
and molecular biology-
1.to separate proteins according to their
electrophoretic mobility.
2.to separate proteins according to their size, and no
other physical feature.

 SDS (the detergent soap) breaks up hydrophobic areas and

coats proteins with negative charges thus overwhelming
positive charges in the protein.
 Therefore, if a cell is incubated with SDS, the membranes
will be dissolved, all the proteins will be solubilized by the
detergent and all the proteins will be covered with many
negative charges.
SDS PAGE
 The denatured Proteins covered negatively charged in the
SDS .
 traveling to the positive since they have negative charge

 These strands go through the tunnel and are separated by

their size.
TRANSFER

 In order to make the proteins accessible to

antibody detection they are moved from within
the gel onto a membrane made of
nitrocellulose or polyvinylidene difluoride
(PVDF) .
 The membrane is placed on top of the gel, and
a stack of filter papers placed on top of that.
 The entire stack is placed in a buffer solution
which moves up the paper by capillary action,
bringing the proteins with it.
 Another method for transferring the proteins is
called electrobloting and uses an electric
current to pull proteins from the gel into
membrane.
WESTERN BLOTTING

 Both varieties of membrane are chosen for

their non-specific protein binding properties
(i.e. binds all proteins equally well).
 Protein binding is based upon hydrophobic
interactions, as well as charged interactions
between the membrane and protein.
 The uniformity and overall effectiveness of
transfer of protein from the gel to the
membrane can be checked by staining the
membrane with coomassie or ponceau S
dyes.
 Nitrocellulose membranes are cheaper
than PVDF, but are far more fragile and do
not stand up well to repeated probings.
 BLOTTING

Blotting used to transfer the samples

from the gel on to a membrane such
as a nylon membrane or
nitrocellulose membrane.
 Analyzed through probing with
nucleic acid probes or antibody
probes.
BLOCKING

 Steps must be taken to prevent interactions between the

membrane and the antibody used for detection of the target
protein (since the antibody is a protein itself).
 Blocking of non-specific binding is achieved by placing the
membrane in a dilute solution of protein.
 Typically Bovin Serum Albumin (BSA) or non-fat dry milk
(both are inexpensive), with a minute percentage of detergent
such as Tween20.
 The protein in the dilute solution attaches to the membrane in all
places where the target proteins have not attached.
 This reduces "noise" in the final product of the Western blot,
leading to clearer results, and eliminates false positives.
 DETECTION

 The membrane is "probed" for the protein of interest with a

modified antibody which is linked to a reporter enzyme,
which when exposed to an appropriate substrate drives a
colourimetric reaction and produces a colour.
Two step
 1•Primary antibody {Antibodies are generated when a host
species or immune cell culture is exposed to the protein of
interest (or a part thereof) }.
 2•A dilute solution of primary antibody (generally between
0.5 and 5 micrograms/mL) is incubated with the membrane
under gentle agitation for anywhere from 30 minutes to
overnight at different temperatures.
 3•The solution is composed of buffered saline solution with
a small percentage of detergent, and sometimes with
powdered milk or BSA.
•Secondary antibody {After rinsing the membrane to remove unbound primary
antibody, the membrane is exposed to another antibody, directed at a species-specific
portion of the primary antibody }.
•The secondary antibody is usually linked to biotin or to a reporter enzyms such as
alkalin phosphatase or horseradish peroxidase. This means that several secondary
antibodies will bind to one primary antibody and enhance the signal.
•Most commonly, a horseradish peroxidase-linked secondary is used to cleave a
chemiluminescent agent, and the reaction product produces luminescence in
proportion to the amount of protein.
•A cheaper but less sensitive approach utilizes a 4-chloronaphthol stain with 1%
horseradish peroxidase; reaction of peroxide radicals with 4-chloronaphthol
produces a dark brown stain that can be photographed without using specialized
photographic film.
ONE STEP

•Historically, the probing process was performed in two steps because of the
relative ease of producing primary and secondary antibodies in separate
processes.

•One-step probing systems that would allow the process to occur faster and with
less consumables.

•This requires a probe antibody which both recognizes the protein of interest
and contains a detectable label, probes which are often available for known
proteins tags.
ANALYSIS

 In practical terms, not all Westerns reveal protein only at

one band in a membrane.
 Size approximations are taken by comparing the stained
bands to that of the marker or ladder loaded during
electrophoresis.
 The process is repeated for a structural protein, such as
actin or tubulin, that should not change between samples.
 This practice ensures correction for the amount of total
protein on the membrane in case of errors or incomplete
transfers.
COLORIMETRIC DETECTION :

 •This method depends on incubation of the Western blot with

a substrate that reacts with the reporter enzyme (such as
peroxidase) that is bound to the secondary antibody.

 •This converts the soluble dye into an insoluble form of a

different color that precipitates next to the enzyme and
thereby stains the membrane.

 •Protein levels are evaluated through densitometry or

spectrophotometry.
DETECTION

RADIOACTIVE DETECTION
 Radioactive labels do not require enzyme substrates, but
rather allow the placement of medical X-ray film directly
against the western blot which develops as it is exposed
to the label and creates dark regions which correspond to
the protein bands of interest.
 Very expensive, health and safety risks are high.
FLUORESCENT DETECTION
 The fluorescently labeled probe is excited by light and the
emission of the excitation is then detected by a photosensor
such as CCD camera.
 Allows further data analysis such as molecular weight
analysis and a quantitative western blot analysis.
 The most sensitive detection methods for blotting analysis.
APPLICATIONS
o The confirmatory HIV test employs a western blot to detect anti-
HIV antibody in a human serum sample. Proteins from known
HIV-infected cells are separated and blotted on a membrane as
above. Then, the serum to be tested is applied in the primary
antibody incubation step; free antibody is washed away, and a
secondary anti-human antibody linked to an enzyme signal is
added. The stained bands then indicate the proteins to which the
patient's serum contains antibody.
 A western blot is also used as the definitive test for Bovine
spongiform encephalopathy (BSE, commonly referred to as 'mad
cow disease').
 Some forms of Lyme disease testing employ western blotting.
 Western blot can also be used as a confirmatory test for Hepatitis
B infection.
 In veterinary medicine, western blot is sometimes used to confirm
FIV+ status in cats.