1. Enzyme Linked Immunosorbent Assay
Dr. Said Sajjad Ali Shah
Research Officer (Parasitology lab)
Veterinary Research Institute, Peshawar
The enzyme-linked immunosorbent assay (ELISA)
is a common laboratory technique which is used to
measure the concentration of an analyte (usually
antibodies or antigens) in solution.
3. History of ELISA
In 1971, Introduced by Peter Perlmann and Eva
Engvall at Stockholm University in Sweden
HIV infection was FIRST
diagnosed through ELISA
4. WHY KNOWN AS ......?
ENZYME LINKED IMMUNOSORBENT ASSAY
Antigen of interest is absorbed on to plastic surface
Antigen is recognized by specific antibody
This antibody is recognized by second antibody
(‘immuno’) which has enzyme attached (‘enzyme-
5. Basic Principle of ELISA
Use an enzyme to detect the binding of antigen (Ag) and
The enzyme converts a colorless substrate (chromogen) to a
colored product, indicating the presence of Ag : Ab binding.
An ELISA can be used to detect either the presence of
Antigens or antibodies in a sample depending how the test
Any molecule that induces production of antibodies when
introduced in the body of an animal is called antigen.
Any “thing”, foreign to the immune system. e.g. bacteria,
viruses, (or their parts), pollen, etc.
Symbol used for Antigen
Proteins produced by the immune system which help
defend against antigens
Each antibody consists of four polypeptides– two
heavy chains and two light chains joined to form a
"Y" shaped molecule
Five different classes of Antibodies
9. Basic Terms
Usually a micro titer plate well
Specially prepared ELISA plates
are commercially available
These have an 8 × 12 well format and can be used with a
wide variety of specialized equipment designed for rapid
manipulation of samples including multichannel pipettes
10. Basic Terms
The process of adding an antigen or antibody,
diluted in buffer, so that it attaches to the solid phase
This is a simple way for immobilization of one of
the reactants in the ELISA and one of the main
reasons for its success
11. Basic Terms
The simple flooding and emptying of the wells with
a buffered solution to separate bound (reacted) from
unbound (unreacted) reagents in the ELISA
12. Basic Terms
An enzyme that is attached irreversibly to a protein,
usually an antibody
A chemical compound with which an enzyme reacts
This reaction is used, in some way, to produce a
signal that is read as a color reaction (directly as a
color change of the substrate or indirectly by its
effect on another chemical)
13. Common Enzymes and substrate
Initially the substrate should be colorless
After degradation by the enzyme it should be strongly colored
ENZYME Substrate Chromogen Stop Solution
Alkaline Phosphatase p-NPP
1 M NaOH
H2O2 Tetramethyl benzidine +
Phosphate – Citrate
1 M H2SO4
H2O2 O – Phenylenediamine +
HCl 1 M HCl
14. Reagents required
Coating Buffer 0.01 M Phosphate Buffer
+ 0.15 M NaCl (PBS)
Diluting/Washing Buffer 0.01 M Phosphate Buffer
+ 0.50 M NaCl + 0.1% Tween 20
Blocking Buffer Bovine Serum Albumin
Enzyme Horse-radish peroxidase
Chromogenic Substrate Trimethyl benzidine
Stop Solution 0.5 M H₂SO₄
20. Types of ELISA
A. Non-Competitive ELISA
1. Direct ELISA
2. In-direct ELISA
3. Sandwich ELISA
B. Competitive ELISA
21. Direct ELISA
An antigen is immobilized in the well of an ELISA
The antigen is then detected by an antibody directly
conjugated to an enzyme such as HRP
Not widely used but common for immuno-
histochemical staining of cells & tissues.
23. Direct ELISA
Quick because only one antibody and fewer steps are used
Cross-reactivity of secondary antibody is eliminated.
Immunoreactivity of the primary antibody might be
adversely affected by labeling with enzymes or tags
Labeling primary antibodies for 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.
25. Indirect ELISA
Then the serum is added, which contains a mixture
of the serum antibodies, of unknown
Some of which may bind specifically to the test
antigen that is coating the well
Incubation of the plate
Washing of the plate
26. Indirect ELISA
Afterwards, a secondary antibody is added, which
will bind to the antibody bound to the test antigen in
This secondary antibody has an enzyme
attached to it
Incubation of the plate
Washing of the plate
27. Indirect ELISA
A substrate for this enzyme is then added.
Often, this substrate changes colour upon reaction
with the enzyme.
The colour change shows that secondary antibody
has bound to primary antibody, which strongly
implies that the donor has had an
immune reaction to the test antigen.
28. Indirect ELISA
Stop solution is used to stop further reaction
The higher the concentration of the primary
antibody that was present in the serum, the stronger
the colour change
Spectrometer/ELISA Reader is used to give
quantitative values for colour strength
29. Interpretation of Results
Percent positivity (PP)
PP= Mean OD of negative control/test sample x 100
Mean OD of positive control
Validity of the test
OD of Positive control ---0.9-2.3
PP of Negative control < 20
(Anaplasma marginale Antibody test, Svanova)
31. Indirect ELISA
A wide variety of labeled secondary antibodies are
Maximum immunoreactivity of the primary antibody is
retained because it is not labeled
Sensitivity is increased because each primary antibody
contains several epitopes that can be bound by the
labeled secondary antibody, allowing for signal
Cross-reactivity might occur with the secondary
antibody, resulting in nonspecific signal
An extra incubation step is required in the procedure.
32. Sandwich ELISA
The procedure for a sandwich ELISA firstly requires the
well of an ELISA plate to be coated with a capture
The analyte or sample is then added, followed by a
The detection antibody can be enzyme conjugated, in
which case this is referred to as a direct sandwich
If the detection antibody used is unlabeled, a secondary
enzyme-conjugated detection antibody is required. This
is known as an indirect sandwich ELISA.
33. Sandwich ELISA
The key advantage of a sandwich ELISA is its high
sensitivity; it is 2-5 times more sensitive than direct or
Sandwich ELISA also delivers high specificity as two
antibodies are used to detect the antigen.
It offers flexibility since both direct and indirect
methods can be used.
If a standardized ELISA kit or tested antibody pair is
not available, antibody optimization has to be worked
out since it is important to reduce cross-reactivity
between the capture and detection antibodies.
35. Competitive ELISA
The known antigen competes for primary antibody
binding sites with the unknown antigen (unknown).
The antibodies are coated on the microplate, then
unknown antigen is applied.
After this the conjugated antigen is applied.
If the sample is in high concentration they will bind
to the antibodies and no binding will occur between
conjugated antigen and antibody.
So the colour will not develop.
38. If the negative controls are giving
Contamination of the substrate solution with
enzyme-labeled antibody, control themselves
Inadequate rinsing of plates
Inadequate blocking of plates
39. If no color has developed for the positive
controls or for the samples:
Check all reagents for dating and storage conditions
Microwell plates not coated properly
Reagents applied in wrong order or step omitted
Enzyme conjugate defective or inhibited by
40. If very little color has developed for positive
controls and the test samples:
Check the dilution of the enzymes labeled antibody
The concentration of the substrate
Wash buffer not adequately drained after every wash step
Inadequate incubation times
Enzyme conjugate defective or inhibited by contaminant,
substrate defective or contaminated
Micro well plates poorly coated
41. If color has developed for the test samples but not
for the positive controls
Check the source of positive controls, their expiry
date and storage
If the color can be seen, but the absorbance is not
high as expected
Check the wave length
1. Always use new tip
3. Plate cover:
During incubation, well
plate should be covered
using the plate cover
45. Advantages of ELISA
Reagents are relatively have long shelf life.
It is highly specific & sensitive (<1pg/ml).
No radiation hazards occur during labeling or disposal of waste.
Easy to perform & quick procedures.
Equipment is widely available.
It can be used to a variety of infections.
It can be used on most type of biological samples like plasma, serum,
urine, cell extracts
It can be used for the diagnosis of carrier animals or chronic infections
46. Disadvantages of ELISA
Measurement of enzyme activity can be more complex than
the measurement of activity of some type of radioisotopes.
Enzyme activity may be affected by plasma constituents.
Kits are not cheap.
Very specific to particular antigen but won’t recognize other
False positive/ negative possible, especially with mutated/