1. Enzyme Linked Immunosorbent Assay
(ELISA)
Dr. Said Sajjad Ali Shah
Research Officer (Parasitology lab)
Veterinary Research Institute, Peshawar

2. ELISA
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
(‘sorbent’)
Antigen is recognized by specific antibody
(‘immuno’)
This antibody is recognized by second antibody
(‘immuno’) which has enzyme attached (‘enzyme-
linked’)

5. Basic Principle of ELISA
 Use an enzyme to detect the binding of antigen (Ag) and
antibody (Ab).
 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
is designed.

6. Antigen
 Any molecule that induces production of antibodies when
introduced in the body of an animal is called antigen.
OR
 Any “thing”, foreign to the immune system. e.g. bacteria,
viruses, (or their parts), pollen, etc.
 Symbol used for Antigen

7. Antibody
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
Solid phase:
 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
Adsorption:
The process of adding an antigen or antibody,
diluted in buffer, so that it attaches to the solid phase
on incubation
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
Washing:
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
 Enzyme conjugate:
An enzyme that is attached irreversibly to a protein,
usually an antibody
Substrate:
A chemical compound with which an enzyme reacts
specifically
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
p-nitrophenyl
phosphatase
p-NPP+
diethandamine+MgCl2
1 M NaOH
Horse radish
Peroxidase
H2O2 Tetramethyl benzidine +
Phosphate – Citrate
buffer
1 M H2SO4
Horse radish
Peroxidase
H2O2 O – Phenylenediamine +
HCl 1 M HCl

14. Reagents required
Reagent Composition
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
(BSA)
Enzyme Horse-radish peroxidase
(HRPO)
Chromogenic Substrate Trimethyl benzidine
(TMB)
Stop Solution 0.5 M H₂SO₄

15. Equipments
Micropipettes
Single channel
Variable ranges (10-200 μL )
Multichannel pipette
An 8-channel 10-200 μL pipette is a good help
for even small-scale work.

16. Equipments
Micro titer plate
Flat bottom Polystyrene plate,
Contain 8 x 12 wells
 Holding capacity 350 μL each

17. Equipments
Washing Device
May be of use particularly when
there is a risk that the samples
tested in ELISA contain
infectious material so must be
collected for subsequent
disinfection

18. Equipments
ELISA Reader

19. ELISA Reader
Principle of the Instrument

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
plate
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.

22. Direct ELISA

23. Direct ELISA
Advantages
 Quick because only one antibody and fewer steps are used
 Cross-reactivity of secondary antibody is eliminated.
Disadvantages
 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.

24. Indirect ELISA
Known antigen is adsorbed to a well in an
ELISA plate
Coated Ag

25. Indirect ELISA
Then the serum is added, which contains a mixture
of the serum antibodies, of unknown
concentration
Some of which may bind specifically to the test
antigen that is coating the well
Incubation of the plate
Washing of the plate
Unbound/Non-
specific Ab
Bound/Specific
Ab

26. Indirect ELISA
Afterwards, a secondary antibody is added, which
will bind to the antibody bound to the test antigen in
the well.
This secondary antibody has an enzyme
attached to it
Incubation of the plate
Washing of the plate
2ndry Ab+Enzyme

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
 PP<25---------------------Negative
 PP>25---------------------Positive
(Anaplasma marginale Antibody test, Svanova)

30. Indirect ELISA

31. Indirect ELISA
Advantages
A wide variety of labeled secondary antibodies are
available commercially
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
amplification
Disadvantages
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
antibody.
The analyte or sample is then added, followed by a
detection antibody.
The detection antibody can be enzyme conjugated, in
which case this is referred to as a direct sandwich
ELISA.
 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
Advantages
The key advantage of a sandwich ELISA is its high
sensitivity; it is 2-5 times more sensitive than direct or
indirect ELISAs.
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.
Disadvantages
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.

34. Sandwich ELISA

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.

37. Troubleshooting
in ELISA

38. If the negative controls are giving
positive results:
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
contaminant

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

42. Precautions
1. Always use new tip
2. Washing:
3. Plate cover:
 During incubation, well
plate should be covered
using the plate cover

43. Precautions
Reagents

44. Precautions
Reagents

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
antigens.
 False positive/ negative possible, especially with mutated/
altered antigen.