3. Immunoblotting techniques include:
1. ELISA
i. Direct ELISA
ii. Indirect ELISA
iii. Sandwich ELISA
iv. Competitive ELISA
2. Western Blotting
3. Southern Blotting
3
4. Introduction:
Blotting:
Visualization of specific DNA,
RNA & protein among many
thousands of contaminating
molecules onto a carrier requires the convergence of
number of techniques which are collectively termed BLOT
transfer.
Immunoblotting:
Immunoblot procedure is an example of modified
technology with much greater specificity.
It detects & analyse individual proteins in complex
mixtures. Involving separation by electrophoresis followed
by staining with antibodies.
4
6. Enzyme linked immuno sorbent assay (ELISA):
ELISA is so named as the test techniques involves an
enzyme system & immunosorbent – an absorbing material
specific for one of the components of reaction, antigen or
antibody.
ELISA is also known as Solid phase enzyme immuno assay
It is usually performed using 96 well microtitre plates
suitable for reading by automization.
A large number of tests can be done at one time.
6
8. Principle:
Most ELISA methods developed for the
detection of antigen or antibody consist if use of
corresponding antibody or antigen which is firmly fixed on
immunosorbent.
The immunosorbent may be particulate.
Enzyme system:
An enzyme is labelled or linked to a specific antigen or
antibody.
Substrate is added to detect the test is +ve or –ve.
Enzyme Substrate
1. Horse radish peroxidase - O-phenyl-diamine-dihydro
chloride.
2. alkaline phosphatase - P-nitrophenyl phosphate
8
9. Mechanism:
The enzyme catalyses (usually hydrolysis) the substrate to
give a colour end point. The intensity of the colour gives
an indication of the amount of bound antibody or antigen.
Alkaline phosphatase – yellow
Horse radish peroxidase – reddish orange
9
11. Direct ELISA:
In direct ELISA, only an
enzyme-labeled primary
antibody is used, meaning
that secondary antibodies
are not needed.
11
12. Indirect ELISA:
In indirect ELISA, both a
primary antibody and a secondary
antibody are used. But in this case,
the primary antibody is not labeled
with an enzyme. Instead, the secondary
antibody is labeled with an enzyme.
12
13. Sandwich ELISA:
Unlike other methods, antigen
coating is not done but coated with
antibody.
13
16. WHY ELISA?
Highly accurate.
Highly sensitive.
Specific, because of the selectivity of the antibody or
antigen.
Just a respective antibody is sufficient.
The plastic Microtitrer plate and the plastic bead are by
far the most convenient. They allow for multiple sample
testing.
16
17. Parameters Advantages Disadvantages Applications
Direct
ELISA
•Simple protocol, time-
saving, and reagents-
saving.
•No cross-reactivity from
secondary antibody.
•High background.
•Low flexibility, since the
primary antibody must be
labeled.
Determining serum
antibody concentrations
(such as with the HIV
test or West Nile virus).
Detection of hepatitis
B markers in serum
Indirect
ELISA
•High flexibility, since the
same secondary antibody
can be used for various
primary antibodies.
•Complex protocol compared
with direct ELISA.
•Cross-reactivity from
secondary antibody.
First screening test
widely used for HIV
because of its high
sensitivity.
Sandwich
ELISA
•High flexibility, High
sensitivity, High
specificity, since different
antibodies bind to the
same antigen for
detection.
•It's sometimes difficult to
find two different
antibodies that recognize
different epitopes on the
antigen of interest and
cooperate well in a
sandwich format.
Detect various kind of
diseases, such
as dengue, malaria,
Chagas disease, Johne's
disease, Detection
of enterotoxin of E.
coli in feces
Competitive
ELISA
High sensitivity, Best for
the detection of small
antigens, even when they
are present in low
concentrations.
•Relatively complex
protocol.
•Needs the use of inhibitor
antigen.
Detection
of Mycobacterium
antibodies in
tuberculosis,Detection
of rotavirus in feces
17
19. Southern blotting:
Sir Edwin Mellor Southern,
Fellow Royal Society and
Trinity won Albert Lasker
Award (2005) in Clinical
Medical Research for the invention of the Southern Blot
in 1975 when he was working as Professor of
Biochemistry at the University of Edinburgh, which is
now a common molecular biology procedure to identify
DNA sequence.
19
20. Introduction:
This method Involves
1. Separation
2. Transfer &
3. Hybridization.
The Southern blot is used to detect the presence of a
particular piece of DNA in a sample.
The DNA detected can be a single gene, or it can be part of
a larger piece of DNA such as a viral genome.
The key to this method is Hybridization.
Hybridization - Process of forming a double-stranded
DNA
molecule between a single-stranded DNA probe and a
single-stranded target patient DNA.
20
21. Steps :
The DNA to be analyzed, such as the total
DNA of an organism, is digested to
completion with a restriction enzyme .
The complex mixture of fragments is
subjected to gel electrophoresis to separate
the fragments according to size.
The restriction fragments present in the gel
are denatured with alkali and transferred onto
a nitrocellulose filter or nylon membrane by
blotting.
This procedure preserves the distribution of
the fragments in the gel, creating a replica of
the gel on the filter.
21
23. Western blotting:
Western blotting is an Immunoblotting technique which rely
on the specificity of binding between a molecule of interest
and a probe to allow detection of the molecule of interest in a
mixture of many other similar molecules.
Introduced by Towbin, et al. in 1979.
In Western blotting, the molecule of interest is a protein and
the probe is typically an antibody raised against that particular
protein.
The SDS-PAGE (Sodium do-decyl sulphate-poly acrylamide
gel electrophoresis) technique is a prerequisite for Western
blotting.
Principle:
It is based on the principle of immunochromatography where
proteins are separated into polyacrylamide gel according to
their molecular weight.
23
24. 1
• Electrophoresing the protein sample
2
• Assembling the Western blot sandwich
3
• Transferring proteins from gel to nitrocellulose paper.
4
• Staining of transferred proteins
5
• Blocking nonspecific antibody sites on the nitrocellulose paper
6
• Probing electroblotted proteins with primary antibody
7
• Washing away nonspecifically bound primary antibody
8
• Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate and
formation of a diaminobenzidine (DAB) precipitate
9
• Photographing the immunoblot
24
26. WHY IMMUNOBLOTTING?
High affinities of antibody toward their epitopes.
It is a very sensitive method and even picogram
quantities of a target protein can be detected.
The two primary advantages of western blotting are
sensitivity and specificity.
Western blotting has advantages over other protein
detection techniques.
26
27. Blotting
techniques
Advantages Disadvantages Applications
Southern
Blotting
•Cheaper than DNA
sequencing.
•Can be used to quantify
the DNA present.
•Used as a definitive
test when identifying
genetically modified
Organisms.
•More expensive than
most other tests.
•Complex and Labor-
intensive.
•Time consuming and
cumbersome
•Requires a large
amount of targeted
DNA.
• Analyze the genetic
patterns which appear in
a person's DNA.
• Used in DNA
fingerprinting, genetic
engineering, & forensic
science for tests such as:
– Paternity testing
– Personal identification
– Sex determination
Western
Blotting
Western blot test is
referred to as the 'Gold
Standard', meaning it
trumps any other
positive tests .
.• Western blotting tells
you how much protein
has accumulated in
cells.
• It is time-consuming
(compared to ELISA)
• If a protein is
degraded quickly,
Western blotting won't
detect it well .
• It might also be more
costly.
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 .
27
28. 1. Gen. procedure of ELISA by DAKO A/S
Produktionsvej 42 • DK-2600 Glostrup • Denmark,
www.dako.com
2. ELISA by Dr. Saba Ahmed M.Phil. Pharmacology
UNIVERSITY OF SARGODHA.
3. Immunoblotting techniques by ; KURGAT GILBERT
IIIBSc. BIOCHEMISTRY.
4. Gordon K, Kochkodan JJ, Blatt H et al. (2013)
Alteration of the EphA2/EphrinA signaling axis in
psoriatic epidermis. J Invest Dermatol 133:712–22
5. Textbook of Microbiology by U.V.Satyanarayana.
6. Textbook of Microbiology by Pelzar.
7. Immunoblotting techniques by Nouf AI-enazi.
8. Science direct & Research gate websites.
28
References:
29. 1. The target molecule type for southern blotting is:
A. RNA.
B. DNA.
C. Protein.
D. Lipids.
2. Limitations of the western blot technique include all of the following except:
A. Low specificity.
B. Loss of antibody epitope with denaturing.
C. Less accurate determination of quantity.
D. Higher cost as compared with ELISA.
3. Which of the following techniques is most commonly employed in modern research?
A. Southern blot.
B. Northern blot.
C. Western blot.
D. Eastern blot.
4. Place the following blotting steps in order:
A. Transfer to membrane.
B. Detect probe.
C. Separate via gel electrophoresis.
D. Treat with probe.
29
QUESTIONIRE:
