ELISA or Enzyme-linked Immunosorbent Assay is a qualitative and quantitative assay for detecting the presence of antigens (virus, hormones, enzymes, etc.) in a sample.

1. S Y E D M U H A M M A D K H A N
B S H O N S . Z O O L O G Y
ELISA: ENZYME-LINKED
IMMUNOSORBENT ASSAY

2. INTRODUCTION
• Immunoassays are biochemical tests that detect
the presence of a given substance (or measure its
concentration) in a solution.
• They use an antibody (usually) or an antigen
(sometimes).
• Two important components:
1. Antigen (Ag)
2. Antibody (Ab)

5. INTRODUCTION
TYPES OF IMMUNOASSAYS: There are several different types of
immunoassays, some of them are:
• Precipitin Test → precipitin (antibodies that precipitate out of a
solution upon binding with an antigen)
• Agglutination Test → agglutination reactions (clumping of cells
due to exposure to specific antibodies). It is used to perform
blood group tests and even in detecting diseases like typhoid
fever, brucellosis, salmonellosis, etc.
• Complement Fixation Test → complement fixing antibodies in the
serum of the subject, which are produced in response to
bacterial antigens.
• Radio-immunoassay (RIA) → radiolabeled molecules. It is used for
the detection of hormones, serum proteins, infectious agents,
vitamins, etc.
• Enzyme-linked Immunosorbent Assay (ELISA) → components of
the immune system (antigens and antibodies) coupled with
enzymes.

6. ENZYME-LINKED IMMUNOSORBENT
ASSAY
• ELISA (Enzyme-linked Immunosorbent Assay) or Enzyme
Immunoassay (EIA) is an immunoassay that uses components
of the immune system (antigens and antibodies) coupled with
enzymes for the detection of immune responses in the body.
• It is so called because:
1. Enzyme-linked: Enzymes are attached with either the
antigen or the antibody.
2. Immune: Antigen is recognized by specific antibody and
sometimes the antibody is recognized by a second one.
3. Immunosorbent: Antigen / antibody of interest is absorbed
on to the plastic surface.
4. Assay: Substrate reacts with enzyme to produce product,
usually colored.

7. HISTORY OF ELISA
• The term ELISA was first used by Engvall & Perlman in
1971.
• First screening test commonly employed for HIV.
• Formerly, the only option for conducting an
immunoassay was radioimmunoassay and as
radioactivity poses a potential health threat, a safer
alternative was sought, i.e. enzymes.
• But the enzyme has to be linked to an appropriate
antibody. This linking process was independently
developed by Stratis Avrameas and G. B. Pierce.

8. HISTORY OF ELISA
• The antibody or antigen has to be fixed to the
surface of the container; i.e., the immunosorbent
must be prepared (Wide and Jerker Porath in 1966).
• In 1971, Peter Perlmann and Eva Engvall at
Stockholm University in Sweden, and Anton Schuurs
and Bauke van Weemen in the Netherlands
independently published papers that synthesized
this knowledge into methods to perform EIA/ELISA.

9. PRINCIPLES OF ELISA
• Antibodies and some antigens can attach to plastic
surfaces.
• Antigens and antibodies can be bonded to
enzymes.
• The enzyme detects the binding of antigen (Ag)
with an antibody (Ab).
• The enzyme converts a colorless substrate
(chromogen) to a colored product.
• An ELISA can be used to detect either the presence
of antigens or antibodies in a sample depending
how the test is designed.

10. APPLICATIONS OF ELISA
• Detection of hormones
• Vaccine quality control
• GMO (Genetically modified organism)
• Detection of infectious agents
• Detection of drug markers
• Detection of immunoglobins
• Detection of tumor markers
• In new born screening
• Detection of serum proteins
• In clinical research

11. SENSITIVITY OF ELISA
• ELISA is one of the most sensitive immunoassays
available.
• It has a typical detection range between 0.01 ng and
0.1 ng.
• Sensitivity relies on the specific characteristics of the
interaction between the antibody and the antigen.
• A number of substrates, like the ones yielding fluorescent
signal or enhanced chemi-luminescent, can be utilized
to enhance results.

12. BASIC STEPS OF ELISA
The following are the basic steps for an ELISA test:
• The sample (antigen / antibody), which may or may not be
labeled, is added to polystyrene micro-titer wells which
already has a reagent (antigen / antibody) bound to its
surface.
• The sample is incubated for a suitable time and at a suitable
temperature to allow binding of the two reagents.
• Excess or unbound reagent is washed off with a washing
buffer solution.
• If the first reagent (antibody) was not tagged then a second
enzyme-linked antibody is also added to bind with the first
one. This second antibody is termed as “antihuman antibody”,
“antiglobulin” or “anti-antibody”. It is also allowed to incubate.

13. BASIC STEPS OF ELISA
• A substrate is added for the enzyme (which may be
bound with either the first or the second reagent
added).
• The enzyme converts the substrate (chromogen) into a
colored substance, or any other type of indicating
signal.
• Importance of Incubation: During the test performance,
incubation time and mentioned temperature is essential
for: (1) proper binding between antigen and antibody,
(2) binding of antibody with the conjugate and (3) color
development of substrate.
• Importance of Washing: It is done to ensure the removal
of any unbound antibody/antigen. Proper washing is
required otherwise the results may become inaccurate.

14. Colored wells of the Micro-titer plate indicates a positive result

15. QUALITATIVE & QUANTITATIVE
ELISA
• Qualitative ELISA: It determines the presence or
absence of antigen or antibody (a simple present /
absent).
• Quantitative ELISA: It determines the quantity of the
antigen or antibody, the optical density (OD) of the
sample is compared to a standard curve, which is
typically a serial dilution of a known-concentration
solution of the target molecule.

16. Standard curve of human IgM at OD 450nm

17. MATERIALS NEEDED FOR ELISA
The following materials are needed to perform an
ELISA test:
1. Testing sample 2. Polystyrene micro-titer plate
3. Pipettes 4. Incubator
5. ELISA reader 6. Antibody (1st & 2nd)
7. Antigen 8. Enzyme
9. Substrate 10. Blocking buffer
11. Stopping solution 12. Washing buffer

18. ELISA test kit for Human IgM

19. MATERIALS NEEDED FOR ELISA
• Specimen Sample For ELISA: serum, cerebrospinal
fluid, sputum, urine, semen, supernatant of culture
stool, etc.
• Polystyrene micro-titer plate: The 96-well plates,
made of polystyrene and are coated with either
inactivated antigen or antibody.
• ELISA reader: It is an instrument used to detect
biological, chemical or physical events of samples
in micro-titer plates.

20. MATERIALS NEEDED FOR ELISA
• Enzyme: Several enzymes are used in ELISA, some of
them are: (1) Horseradish peroxidase (most commonly
used), (2) Alkaline phosphatase, (3) β-galactosidase, (4)
Lacto-peroxidase and (5) Tetra-methyl benzidine.
• In case of peroxidase, the substrate hydrogen peroxide
is converted into water and O2 in the presence of
electron donors, i.e. diaminobenzidine or 4-
chloronaphthol, etc.
• The electron donors are themselves oxidized.
• Oxidation of diaminobenzidine produces dark brown
color while that of 4-chlorornaphthol yields purple color
which is the basis of ELISA

21. MATERIALS NEEDED FOR ELISA
• Substrate: Initially colorless but strongly colored or
fluorescent after enzyme action.
• Blocking buffer: It is a solution of irrelevant protein,
mixture of proteins, or other compound that
passively adsorbs to all remaining binding surfaces
of the plate.
• Stopping solution: It stops the enzyme substrate
reaction and color development.
• Washing buffer: It acts as a buffered solution
containing detergent to wash unbound material
from the plate.

22. Polystyrene Micro-titer Plate (96 wells)

23. ELISA
Reader

24. TYPES OF ELISA
• There are five basic variants of ELISA test, these are:
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
5. Reverse ELISA

25. DIRECT ELISA
• Direct ELISA is a type of ELISA in which only a labeled
primary antibody (bound with an enzyme) is used to
detect the presence of an antigen.
• It has the following steps:
• Coating surface with antigens: A buffered solution of the
antigen to be tested is added to each well (usually 96-
well plates) of a micro-titer plate.
• Adhesion: It is given time to adhere to the plastic
through charge interactions.
• Blocking unoccupied sites: A solution of non-reacting
protein, such as bovine serum albumin or casein, is
added to each well in order to cover any plastic surface
in the well which remains uncoated by the antigen.
• Washing: Excess or unbound antigens are washed off.

26. DIRECT ELISA
• Addition of primary antibody: The primary antibody with
an attached (conjugated) enzyme is added, which
binds specifically to the test antigen coating the well.
• Washing: Excess or unbound antibodies are washed off.
• Addition of substrate and color change: A substrate for
this enzyme is then added, which changes color upon
reaction with the enzyme. The higher the concentration
of the primary antibody in the serum, the stronger the
color change.
• Quantification via spectrometry: Often, a spectrometer is
used to give quantitative values for color strength.

27. Basic steps and mechanism of direct ELISA

28. DIRECT ELISA
Advantages
• It is a short protocol.
• It saves time and reagents.
• There is no cross-reactivity
from secondary antibody.
Disadvantages
• The method of antigen
immobilization is not
specific; when serum is used
as the source of test
antigen, all proteins in the
sample may stick to the
micro-titer plate well.
• Small concentrations of
analyte (antibody or
antiserum) in serum must
compete with other serum
proteins when binding to
the well surface.

29. INDIRECT ELISA
• A primary, unlabeled antibody binds with immobilized
antigen and is in turn bound with a secondary enzyme
linked antibody, which will give color change upon the
addition of a suitable substrate. It is used to test for
antibodies.
• It has the following steps:
• Coating surface with antigens: The polystyrene micro-
titer wells are coated with antigens.
• Washing: Excess or unbound antigens are washed off.
• Addition of primary antibody: Test antiserum (containing
primary unlabeled antibody) is added and allowed to
incubate.

30. INDIRECT ELISA
• Binding of antigen and primary antibody: The primary
antibody will bind with the antigen (given that it was
present in the antiserum).
• Washing: Excess or unbound primary antibodies are
washed off.
• Addition of secondary antibody: A secondary enzyme-
linked antibody is added which binds with the primary
antibody.
• Washing: Excess or unbound antibodies are washed off.
• Addition of substrate and color change: The substrate for
the enzyme is added and a color change is observed
due to the action of the enzyme.

32. INDIRECT ELISA
Advantages
• It exhibits signal
amplification, i.e.
several secondary
antibodies will bind to
the primary antibody.
• It has a high flexibility,
i.e. the same
secondary antibody
may be used for
several primary
antibodies.
Disadvantages
• It is a long protocol if
compared to direct
ELISA.
• There is a possibility of
cross-reactivity from
secondary antibody.

33. SANDWICH ELISA
• The antigen is sandwiched between a capture
antibody (bound to the well) and the primary
antibody which is added later on.
• The secondary antibody, which is labeled (bound
with enzyme) then binds with the primary antibody
and after the addition of substrate.
• It can be both direct and indirect.

34. Basic steps and mechanism
of Direct Sandwich ELISA

35. Basic steps and mechanism
of Indirect Sandwich ELISA

36. SANDWICH ELISA
Advantages
• High specificity: two antibodies
detecting different epitopes on the
same antigen.
• It is suitable for complex samples.
• It has a high flexibility and
sensitivity, i.e. both direct and
indirect methods can be used.
• Use of the purified specific
antibody to attach the antigen to
the plastic eliminates a need to
purify the antigen from
complicated mixtures before the
measurement.
Disadvantages
• It has a demanding design.
• Finding two antibodies against the
same target that recognize
different epitopes and work well
together can be challenging at
times.

37. COMPETITIVE ELISA
• In this test, a competition is generated between the
antigens (between those bound to the micro-titer well
and those which are already bound with the antibody).
• Its steps are as follows:
• Incubation of antibody and its antigen: Unlabeled
antibody is incubated in the presence of its antigen
(sample).
• Addition of the antigen-antibody complex: These bound
antibody-antigen complexes are then added to an
antigen-coated well.
• Competition: The more antigens in the sample, the more
antigen-antibody complexes are formed and so there
are less unbound antibodies available to bind to the
antigen in the well, hence "competition".

38. COMPETITIVE ELISA
• Washing: The plate is washed, so unbound
antibodies are removed.
• Addition of secondary antibody: The secondary
antibody (enzyme-linked), specific to the primary
antibody, is added.
• Washing: Excess or unbound secondary antibodies
are washed off.
• Addition of substrate: A substrate is added which
produces a color (chromogenic) or fluorescent
signal (due to the activity of the enzyme).

39. Basic steps and mechanism of competitive ELISA

40. COMPETITIVE ELISA
Alternative form of Competitive ELISA:
• Some competitive ELISA kits include enzyme-linked
antigen rather than enzyme-linked antibody.
• The labeled antigen competes for primary antibody
binding sites with the sample antigen (unlabeled).
• The less antigen in the sample, the more labeled
antigen is retained in the well and the stronger the
signal.
• Commonly, the antigen is not first positioned in the
well.

41. REVERSE ELISA
• This test leaves the antigens suspended in the test
fluid from which all of the excess antibodies are
pulled out by “scavenger antigens” and the
remainder solution is passed through a detector for
analysis.
• It has the following steps:
• Incubation of unlabeled antibody with the sample
(antigen): Unlabeled antibody is incubated in the
presence of its antigen (sample). Sufficient
incubation period is provided to allow the
antibodies to bind to the antigens.

42. REVERSE ELISA
• Passing through scavenger channel: The sample is then
passed through a “scavenger channel” which is a test
tube or a specifically designed flow through channel
that has “scavenger antigens” bound to its surface.
These can be identical or sufficiently similar to the
primary antigens that the free antibodies will bind.
• Binding with excess antibodies: The scavenger antigens
bind will all the excess antibodies introduced into the
sample, if proper time and surface area is provided.
• Detector: The sample, that now contains the tagged
and bound antibodies, is passed through a detector. This
device can be a flow cytometer or other device that
illuminates the tags and registers the response.

43. REVERSE ELISA
Advantages
• Allows multiple antigens to
be tagged and counted at
the same time.
• This allows specific strains of
bacteria to be identified by
two (or more) different color
tags. If both tags are
present on a cell, then the
cell is that specific strain. If
only one is present, it is not.
• The equipment needed is
usually less complicated
and can be used in the
field.
Disadvantages
• This test is done, generally,
one test at a time and
cannot be done with the
micro-titer plate.

44. ADVANTAGES OF ELISA
• Reagents are relatively cheap.
• Reagents have a long shelf life.
• ELISA is highly specific and sensitive.
• No radiation hazards occur during labeling or
disposal of waste.
• Easy to perform.
• Procedures are quick (not very time consuming).
• Home kits are inexpensive and widely available.
• ELISA can be used to detect a variety of infections.

45. DISADVANTAGES OF ELISA
• Measurement of enzyme activity can be more complex
than measurement of activity of some type of
radioisotopes.
• Enzyme activity may be affected by plasma
constituents.
• Kits are commercially available, but not cheap.
• Very specific to a particular antigen, i.e. it won’t
recognize any other antigen.
• False positive/negative results are possible, especially
with mutated/altered antigen.
• Results may not be absolute.
• Concentration may be unclear.
• Appropriate antibodies must be available.

46. FINAL VERDICT ON ELISA
ELISA tests have their drawbacks and flaws that may
overshadow their merits at some points but overall
the tests are very practical and are pretty popular as
well ranging from simple house-hold pregnancy test
kits to complex blood profile tests (HIV, hepatitis,
Mycobacterium, etc.)