ELISA

&
WESTERN BLOTTING
BIOMEDICAL DIAGNOSTICS
ELISA (enzyme-linked
immunosorbent assay)
• A plate-based assay technique designed for detecting
and quantifying peptides, proteins, antibodies and
hormones.
• In an ELISA, an antigen must be immobilized to a solid
surface and then complexed with an antibody that is
linked to an enzyme.
• Detection is accomplished by assessing the conjugated
enzyme activity via incubation with a substrate to
produce a measurable product.
• The most crucial element of the detection strategy is a
highly specific antibody-antigen interaction.
Characteristic Advantages of ELISA
Typically performed in 96-well (or 384-well) polystyrene
plates, which will passively bind antibodies and proteins –
many samples can be evaluated at the same time

It is this binding and immobilization of reagents that makes

ELISAs so easy to design and perform.

Having the reactants of the ELISA immobilized to the

microplate surface makes it easy to separate bound from
non-bound material during the assay.

The ability to wash away nonspecifically bound materials

makes the ELISA a powerful tool for measuring specific
analytes within a crude preparation.
General ELISA Procedure:
Antigen on surface
Step 1. A coating step: the first layer, consisting of a target antigen or antibody,
is adsorbed onto a 96-well polystyrene plate.

Step 2. A blocking step: All unbound sites are coated with a blocking agent.

Step 3: A series of Washing steps

Step 4. An incubation step: the plate is incubated with enzyme-conjugated

antibody.

Step 5. A series of Washing steps: removes all unbound antibody.

Step 6. An addition step: A substrate is then added, producing a calorimetric

signal.

Step 7. The final step: the plate is read in terms of the signal produced; multiple
layers of antibodies can be used to amplify signal in detection.
Types of ELISA
• Types: direct, indirect, sandwich or competitive.
• The key step, immobilization of the antigen of interest, can be
accomplished by:
• direct adsorption to the assay plate, or
• indirectly via a capture antibody that has been attached to the plate.
• The antigen is then detected by:
• directly (enzyme-labeled primary antibody) or
• indirectly (enzyme-labeled secondary antibody)
• The detection antibodies are usually labeled with:
• alkaline phosphatase (AP) or
• horseradish peroxidase (HRP).
• A large selection of substrates is available for performing the ELISA with
an HRP or AP conjugate. The choice of substrate depends upon:
• required assay sensitivity and
• instrumentation available for signal-detection (spectrophotometer,
fluorometer or luminometer).
 1. Direct ELISA:
An antigen coated to a multi-well plate is detected by an
antibody that has been directly conjugated to an enzyme

Advantage Disadvantage

• Quick because only one • Immunoreactivity of the

antibody and fewer steps primary antibody might be
are used. adversely affected by labeling
• Cross-reactivity of with enzymes or tags.
secondary antibody is • Labeling primary antibodies for
eliminated. each specific ELISA system is
time-consuming and expensive.
• No flexibility in choice of
primary antibody label from one
experiment to another.
• Minimal signal amplification.
 2. Indirect ELISA:
An antigen coated to a multi-well plate is detected by an
antibody that has been directly conjugated to an enzyme
Advantage Disadvantage

• A wide variety of labeled secondary antibodies • Cross-reactivity

are available commercially. might occur with
• Versatile because many primary antibodies can the secondary
be made in one species and the same labeled antibody, resulting
secondary antibody can be used for detection. in nonspecific
• Maximum immunoreactivity of the primary signal.
antibody is retained because it is not labeled. • An extra incubation
• Sensitivity is increased because each primary step is required in
antibody contains several epitopes that can be the procedure.
bound by the labeled secondary antibody,
allowing for signal amplification.
 3. Sandwich ELISA :
-uses matched antibody pairs, where each antibody is specific
for a different, non-overlapping part (epitope) of the antigen
molecule.
- A first antibody (known as capture antibody) is coated to the
wells.
- The sample solution is then added to the well.
- A second antibody (known as detection antibody) follows
this step in order to measure the concentration of the sample
Advantage Disadvantage
• High specificity: the antigen/analyte is specifically • Cost
captured and detected • Time and no. of
• Suitable for complex (or crude/impure) samples: steps/ complexity
the antigen does not require purification prior to
measurement
• Flexibility and sensitivity: both direct or indirect
detection methods can be used
4. Competitive/inhibition ELISA :
-competitive reaction between the sample antigen and
antigen bound to the wells of a microtiter plate with the
primary antibody
A. Primary antibody is incubated with the sample antigen
B. Resulting antibody-antigen complexes are added to wells
that have been coated with the same antigen.
C. After an incubation period, any unbound antibody is
washed off.
- The more antigen in the sample, the more primary
antibody will be bound to the sample antigen.
Advantage Disadvantage
• high sensitivity to compositional • there will be a smaller amount of
differences in complex antigen primary antibody available to bind
mixtures, even when the specific to the antigen coated on the well,
detecting antibody is present in resulting in a signal reduction
relatively small amounts
Diagnostic Tests
Detect and Measure the Presence of Antibodies in the
Blood
• Autoantibodies (anti-dsDNA, anti-dsg1, ANA, etc.)
• Antibodies against infectious disease (antibacterial, antiviral, antifungal)
• Hepatitis A, B, C, HIV, etc.
• hepatitis B virus surface antigen (HBsAg)
• Anit-HIV type 1 (group M - O) or type 2

Detect and Estimate Detect and Estimate Hormone

the Levels of Tumor Levels
Markers • Luteinizing hormone
• Prostate-specific antigen • Follicular stimulating hormone
(PSA) • Prolactin
• Carcinoembryonic Antigen • Testosterone
(CEA) • Human chorionic gonadotropin (hCG)
Diagnostic Tests
Tracking Disease Outbreaks Detecting Past Exposures
• Cholera • HIV
• HIV • Lyme disease
• Influenza • Hepatitis

Screening Donated Blood Detecting Drug Abuse

for Possible Viral • Amphetamine
Contaminants • Methamphetamine
• Anti-HIV-1/2 • 3,4-methylenedioxy-
• Anti-HCV methamphetamine
• HBsAg • Cocaine
• Benzoylecgonine
Western
Blotting
 Basic
Procedure
Principle of WB
• Western blotting separates, detects, and identifies one or more
proteins in a complex mixture.
• It involves separating the individual proteins by polyacrylamide
gel electrophoresis (SDS-PAGE) and then transferring or blotting
onto an overlying strip of nitrocellulose or nylon membrane by
electro-blotting.
• Once the proteins are in the membrane, they can be detected
using antibodies labeled with probes, such as radioactive
isotopes or enzymes.
• When such probes are used, the detection limits can be 10 to
100 times lower (i.e. higher sensitivity) than when direct
immunoprecipitation and staining of proteins are conducted.
• Quantifying bands on a western blot by densitometry allows a
researcher to quantitatively compare samples (e.g., a treatment
or time effect).
Interfering Factors
Limitations:
• Western blot is a very delicate and time-consuming process.
A minute imbalance at any level of the procedure can skew the
results of the entire process.
• The secondary antibody can sometimes react with a non-
intended protein, and this can cause the labeling of an
incorrect protein.
• Insufficient transfer time can result in the larger proteins
not transferring correctly. This can cause erroneous bands or
no bands at all.
• Well-trained technicians are a must for this technique.
• Western blot is semi-quantitative at best. Only an
approximate estimation and not a precise measurement of the
molecular weight of the protein is possible.
• Primary antibody availability is crucial. If a primary
antibody is not available for a specific protein, western blotting
cannot be used to detect that protein.
Clinical significance of WB:
• WB is used to detect anti-HIV antibodies in human
serum and urine samples. It is usually performed after
the ELISA test to confirm the diagnosis of HIV. It is
far more sensitive than the ELISA test.
• WB is also useful in detecting Lyme disease and
atypical and typical bovine spongiform
encephalopathy.
• When determining cancers, incongruous isoforms of
proteins can become potential markers of the pathology
of the disease.
• Autoantibodies may also indicate an autoimmune
disease.
• To detect: protein-DNA interactions, protein-protein
interactions, post-translational modifications (PTMs),
protein isoform detection, antibody characterization,
epitope mapping, and subcellular protein localization.
Thank you!