Brief presentation on Western Blotting and ELISA

1. Western Blotting & ELISA
By
Majid KB
Adnan Khan
Anissullah
Malik hamza
Irfan Khan
Fazal
Imran

2. Western blotting
• The Western blot is an analytical technique used to detect
specific proteins in a given sample of tissue homogenate or
extract.
&
• Immunoblotting: because an antibody is used to specifically
detect its antigen
• Western blot for Proteins was Developed by George Stark
using antibodies to locate Proteins

3. Western blotting
• Western blotting is based on building antibody-protein complex
through specific binding of antibodies to proteins immobilized on a
membrane.
• Then detecting the bound antibody through several detection
methods

4. The Western blotting workflow
• Sample preparation
• Gel electrophoresis
• Transfer
• Blocking & Antibody probing
• Detection
• Analysis

5. Sample preparation
• Protein from all sources can be used for western blotting such as from
single cell, whole tissues, extracellular matrices, biological fluids.
• Protein can be extracted from the sample in three distinct steps:
• Cell lysis: Physical method (ultra sonication, homogenization etc.)
Chemical method (use of detergents)
Biological method (enzymatic digestion)
• Protein isolation from the rest of cellular components:
Centrifugation, DNase and other lysis buffer is used for the
removal of cell organelles, nucleic acids, polysaccharides etc.
• Protein purification:

6. Gel Electrophoresis
• PAGE (Polyacrylamide gel electrophoresis) is mainly used for the
separation of protein particles.
• SDS is added to the gel and running buffer to denature the protein
molecules which will ensure that your proteins are separated solely
on the basis of size and not on three-dimensional structure.
• Molecular weight markers are used to define the size of proteins run
in a gel. Markers are composed of different proteins of known size.
• As proteins are not directly visible in the gel, the gel must be stained.
Proteins are usually stained with dyes such as Blue, Silver stain, or
Deep Purple.

7. Protein Transfer
• Protein are transferred from the gel to a solid support membrane
such as nitrocellulose or polyvinylidene difluoride (PVDF).
• The proteins transferred from the gels are immobilized which makes it
possible to detect the proteins on the membrane using specific
antibodies.
• Electro-blotting is mainly used which uses electric field to pull
proteins from the gel onto the PVDF or nitrocellulose membrane.
• The proteins move from within the gel onto the membrane while
maintaining the organization they had within the gel

9. Blocking &Antibody Probing
• Western blotting involves the immobilization of biomolecules on a
membrane and blocking the spaces not already occupied by proteins.
• As non-specific binding of antibodies to the membrane reduces the
specificity and sensitivity of the assay.
• Two main classes of blocking agents (proteins and non-ionic
detergents) are commonly used for western blotting.

10. Antibody Probing
• Western blotting protocols usually uses a non-labeled primary
antibody directed against the target protein and labeled secondary
antibody directed against the constant region of the primary
antibody.
• The secondary antibody serves not only as a carrier of the label but is
also a mechanism to amplify the emitted signals because secondary
antibodies are most often polyclonal and can bind primary antibody
at different epitopes simultaneously.

11. Antibody Probing
• Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the
two most commonly used enzymes for protein detection using
chromogenic substrates.
• Other antibody probes used for protein detection are:
• Fluorophores
• Biotinylated secondary antibodies
• Radioisotopes.

12. Detection
• Different detection system are used in western blotting based on
fluorescence, chromogenic or radio-isotopic detection.
• Enzymatic detection methods require the addition of a substrate that
emits light when it reacts with an enzyme conjugated to a secondary
antibody.
• Fluorescence-based detection requires the excitation of fluorophore
using a light source of a specific wavelength causing light emission.

13. Chemiluminescence Fluorescence-based detection

14. Analysis
• Detection of signals, using either X-
ray film, scanners or a charge-
coupled device (CCD) camera-based
imager, results in image containing
visible protein bands.
• the presence and the amount of a
protein of interest is estimated by
visual analysis, and the size can be
determined by comparison to a
known molecular weight marker
Molecular
weight
markers 1 2 3 4
Mr 102 000
Mr 76 000
Mr 52 000

15. Applications of Western Blotting
• Diagnosis of HIV by ELISA involves the western blotting technique.
• Western blotting technique is also used to detect some forms of Lyme
Diseases.
• Western blotting technique is used in definitive test for BSE.
• Confirmatory test for Hepatitis-B involves western blotting technique.
• Western blotting test is used in the analysis of Biomarkers such as
hormones, growth factors and cytokines.
• This technique is also employed in The Gene expression studies.

16. Limitations of Western Blotting
• Very delicate and time consuming process. A minute imbalance at any
level of the procedure can skew the results of entire process.
• Cause erroneous in bands or no bands due to insufficient transfer.
• Well trained techniques are required for this technique.
• Primary antibody availability is crucial.
• It is just a Semi-Quantitative test. Only estimation and not a precise
• measurement of molecular weight of the protein is possible.

17. ELISA

18. Introduction
• The enzyme-linked immuno sorbent assay (ELISA) is an assay technique
designed for detecting and quantifying peptides, proteins, antibodies and
hormones.
• The ELISA has been used as a diagnostic tool in medicine and plant
pathology, as well as a quality-control check in various industries.
• The process of the ELISA result in a colored end product which correlates
to the amount of analyte present in the original sample.

19. Introduction
• ELISAs are typically performed in 96 well (or 384 well) polystyrene
plates.

20. Principle
• Principle of Enzyme-linked Immunosorbent Assays (ELISAs) is based on
the specific interaction between antibody and antigen.
• The ELISA can be used to detect antigens that are recognized by an
antibody or it can be used to detect antibodies that are recognized by an
antigen.

21. ELISA Procedure

22. Antibodies for ELISA
• All ELISA formats rely on the specific
interaction between an antigen and
a matching antibody.
• The antibodies used in an ELISA can
be either:
• monoclonal (capable of specific
binding to a single unique epitope)
• OR
• polyclonal (capable of binding to
multiple epitopes).

23. Probes for ELISA
• Radioactive isotopes, enzymes and fluorescent dyes
are different types of chemical tags that have been
conjugated to secondary or primary
antibodies(probes) to make probes detectable.
• Horseradish peroxidase (HRP) is most commonly used
for blotting, immunoassays and it converts its
substrate ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-
6-sulfonic acid]-diammonium salt) in green end
product.

24. Blocking & Washing Buffers
• A blocking buffer is a solution of irrelevant protein, with a small
percentage of detergent. Blocking buffer block the places where
target proteins have not been attached.
• Washing buffer are used to remove nonspecifically bound and
unbound reagents.
• Washing is performed by buffers such as Tris-buffered saline (TBS) or
phosphate-buffered saline (PBS) with detergent such as 0.05% Tween-
20.

25. Types of ELISA
• INDIRECT ELISA
• DIRECT ELISA
• SANDWICH ELISA
• COMPETETIVE ELISA
NON -COMPETETIVE
ELISA

26. Indirect ELISA
• Antigen is added to plate.
• Added Blocking buffer.
• Suitable primary antibody is added.
• Secondary antibody- HRPO is then added
which recognizes and binds to primary
antibody.
• TMB substrate is added, is converted to
detectable form.

27. Advantages of Indirect Detection
• Wide variety of labeled secondary antibodies are available commercially.
• Sensitivity is increased because each primary antibody contains several
epitopes that can be bound by the labeled secondary antibody, allowing
for signal amplification.
Disadvantages of Indirect Detection
• Cross-reactivity may occur with the secondary antibody, resulting in
nonspecific signal.
• An extra incubation step is required in the procedure.

28. Direct ELISA
• Apply a sample of known antigen to a surface.
• Enzyme linked primary antibody is applied to the
plate.
• Wash the plate. After this wash, only the
antibody-antigen complexes remain attached.
• Apply a substrate which is converted by the
enzyme to elicit a chromogenic signal.

29. Advantages of Direct Detection
• Quick methodology since only one antibody is used.
• Cross-reactivity of secondary antibody is eliminated.
Disadvantages of Direct Detection
• Labeling of every primary antibody is time-consuming and
expensive.
• Little signal amplification.

30. Sandwich ELISA
• Plate is coated with suitable antibody.
• Blocking buffer is added.
• Sample is added to plate so antigen is bounded by capture antibody.
• A suitable biotin labeled detection antibody is added to plate.
• Enzyme HRPO is added and binds the biotin labeled detection antibody.
• TMB substrate is added and converted by HRPO to colored product.

31. Competitive ELISA
• Solid phase coated
with antibody
• Add unknown amount of
unlabeled antigen and known
amount of labeled antigen
• Free and labeled antigen are
captured
• Color formation by oxidation of
substrate into a colored
compound

32. Advantages
• Suitable for complex samples, since the antigen does
not require purification prior to measurement.
Disadvantages
• Each antigen may require a different method to couple
it to the enzyme.

33. • Presence of antigen or the presence of antibody in a sample can
be detected by ELISA.
• ELISA can be used to determination of serum antibody
concentrations in a virus test (such as HIV test).
• ELISA can also be used in home pregnancy test.
• It is used in food industry to detect potential food allergens such
as milk, peanuts, walnuts, almonds, and eggs.
• ELISA can also be used in toxicology as a rapid presumptive
screen for certain classes of drugs.
Applications of ELISA

34. • A positive result correctly confirming the presence of an antibody
does not necessarily mean the patient is sick. The body can continue
to produce antibodies even though the person may have had the
disease earlier and recovered.
• People may be poor producers of an antibody or may have some
interfering substance in their blood. The amount of antibody,
consequently, may be too low to measure accurately or may go
undetected. This result is termed a false negative.
Limitations of ELISA

35. • Western blotting and Enzyme-linked immunosorbent assay (ELISA)
are both based on the specific interaction between antigen and
antibody.
• They are widely used as diagnostic tools in medicine and as quality
control measures in various industries.
• They also used as analytical tools in biomedical research for the
detection and quantification of specific antigens or antibodies in a
given sample.
Conclusion