ELISA: Enzyme-linked Immunosorbent Assay

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.

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ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
Introduction
Immunoassays are biochemical tests that detect the presence of a given substance (or
measure its concentration) in a solution through the use of an antibody (usually) or an antigen
(sometimes).
Antigen (Ag) is any molecule that induces production of antibodies when introduced in the
body of an organism (any-thing foreign to the immune system), i.e. Bacteria, Viruses (or their
parts), etc. Antibody (Ab) is a protein produced by the immune system in response to an
antigen to defend the organism against it. They are also termed as immunoglobins.

Figure: Antigens induce an immune system response by interacting with an antibody that
matches the antigen's molecular structure.
Enzyme-linked Immunosorbent Assay (ELISA)
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.
ELISA has a high sensitivity and is very useful in estimating ng/mL to pg/mL of the required
substance in the solution. The following are the major principles involved in an ELISA test:

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 Antibodies and some antigens can attach to plastic surfaces and still maintain their full
immunological capabilities.
 Antigens and antibodies can be bonded to enzymes and the resulting complexes are still
fully functional.
 ELISA uses an enzyme to detect the binding of antigen (Ag) with an antibody (Ab).
 The enzyme converts a colorless substrate (chromogen) to a colored product, indicating
the presence of antigen-antibody binding.
 An ELISA can be used to detect either the presence of antigens or antibodies in a sample
depending on how the test is designed.
Applications
 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 newborn screening
 Detection of serum proteins
 In clinical research

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. ELISA sensitivity relies on the specific characteristics of the
interaction between the antibody and the antigen. Additionally, several substrates, like the
ones yielding fluorescent signal or enhanced chemiluminescent, can be utilized to enhance
results.
Generalized 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 the
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.
 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 the antibody with the conjugate, and (3) color development of substrate.

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 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.
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.
Materials Needed
The following materials are needed to perform an ELISA test:
 Testing sample
 Polystyrene microtiter plate
 Pipettes
 Incubator
 ELISA reader
 Antibody (1
st
& 2
nd
)
 Antigen
 Enzyme
 Substrate
 Blocking buffer
 Stopping solution
 Washing buffer
Specimen Sample For ELISA: serum, cerebrospinal fluid, sputum, urine, semen, the
supernatant of culture stool, etc.
Polystyrene microtiter plate: The 96-well plates are made of polystyrene and are coated with
either inactivated antigen or antibody. The function of the plate has to hold the immobilized
either antigen or antibody. Antigen or antibody present in the sample will bind to the plate.

Figure: A 96-well polystyrene microtiter plate.
ELISA reader: It is an instrument used to detect biological, chemical, or physical events of
samples in microtiter plates. It is widely used in research, drug discovery, bioassay validation,
quality control, and manufacturing processes in the pharmaceutical and biotechnological
industry and academic organizations.

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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 the 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. which are themselves oxidized. Oxidation of diaminobenzidine produces
dark brown color while that of 4-chlorornaphthol yields purple color which is the basis of
ELISA
Substrate: Initially the substrate should be colorless but after degradation by the enzyme it
should be strongly colored or fluorescent.
Blocking buffer: It is a solution of irrelevant protein, a mixture of proteins, or other
compounds that passively adsorbs to all remaining binding surfaces of the plate. The blocking
buffer is effective if it improves the sensitivity of an assay by reducing background
interference, i.e. bovine serum albumin or casein.
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.
Types of ELISA & Their Procedures
There are five basic variants of ELISA tests, these are: (1) Direct ELISA, (2) Indirect ELISA, (3)
Sandwich ELISA, (4) Competitive ELISA, and (5) Reverse ELISA. Their details are as follows:
1. 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 to cover any plastic surface in the well which
remains uncoated by the antigen.
 Washing: Excess or unbound antigens are washed off.
 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.

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 Quantification via spectrometry: Often, a spectrometer is used to give quantitative
values for color strength.

Figure: Direct ELISA.
Advantages of Direct ELISA: It is a short protocol, it saves time and reagents. There is no cross-
reactivity from the secondary antibody.
Disadvantages of Direct ELISA: A major disadvantage of the direct ELISA is 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 microtiter plate wells, so small concentrations of
analyte (antibody or antiserum) in serum must compete with other serum proteins when
binding to the well surface.

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2. Indirect ELISA: Indirect ELISA is a type of ELISA test in which 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 microtiter 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.
 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.
Advantages of Indirect ELISA: 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 of Indirect ELISA: It is a long protocol if compared to direct ELISA. There is a
possibility of cross-reactivity from the secondary antibody.
3. Sandwich ELISA: Sandwich ELISA is a type of ELISA in which 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, the antigen-antibody binding is
detected via color appearance. It can be used to detect both: antigens and antibodies. It
can be both direct and indirect. It has the following steps:
1. Addition of capture antibody: A surface is prepared to which a known quantity of
“capture antibody” (which is meant to trap a specific antigen) is bound.
2. Blocking unoccupied sites: Any nonspecific binding sites on the surface are blocked.
3. Addition of sample containing antigen: The antigen-containing sample is applied to the
plate, and captured by an antibody.
4. Washing: The plate is washed to remove unbound antigen.
5. Addition of primary antibody: A specific antibody (primary) is added which binds to the
antigen (sandwich: the antigen is stuck between two antibodies). In the case of a
direct Sandwich ELISA, this antibody is an enzyme-linked, whereas, in indirect

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technique, another enzyme-linked antibody must be added to bind with the first
antibody.
6. Addition of secondary antibody: Enzyme-linked secondary antibodies are applied as
detection antibodies that bind specifically to the primary antibody’s non-specific
region.
7. Washing: The plate is washed again to remove the unbound antibody-enzyme
conjugates.
8. Addition of substrate: A substrate is added to be converted by the enzyme into a color
or fluorescent or electrochemical signal.
9. Quantification: The absorbance or fluorescence or electrochemical signal (e.g.,
current) of the plate wells is measured to determine the presence and quantity of
antigen.

Figure: Sandwich ELISA, indirect variant.
Advantages of Sandwich ELISA: It has a high specificity because it involves two antibodies
detecting different epitopes (the part of an antigen molecule to which an antibody attaches
itself) 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. Without the first layer of
"capture" antibody, any proteins in the sample (including serum proteins) may competitively
adsorb to the plate surface, lowering the quantity of antigen immobilized. 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, simplifying the assay, and increasing
the specificity and the sensitivity of the assay.
Disadvantages of Sandwich ELISA: It has a demanding design, i.e. finding two antibodies
against the same target that recognizes different epitopes and works well together can be
challenging at times.
4. Competitive ELISA: In this test, a competition is generated between the antigens
(between those bound to the microtiter well and those which are already bound with the
antibody). Its steps are as follows:

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1. Incubation of antibody and its antigen: Unlabeled antibody is incubated in the
presence of its antigen (sample).
2. Addition of the antigen-antibody complex: These bound antibody-antigen complexes
are then added to an antigen-coated well.
3. 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".
4. Washing: The plate is washed, so unbound antibodies are removed.
5. Addition of secondary antibody: The secondary antibody (enzyme-linked), specific to
the primary antibody, is added.
6. Washing: Excess or unbound secondary antibodies are washed off.
7. Addition of substrate: A substrate is added which produces a color (chromogenic) or
fluorescent signal (due to the activity of the enzyme).
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.
Detection of HIV via Competitive ELISA: For the detection of HIV antibodies, the wells of
microtiter plates are coated with the HIV antigen. Two specific antibodies are used, one
conjugated with enzyme and the other present in serum (if serum is positive for the
antibody). Cumulative competition occurs between the two antibodies for the same antigen,
causing a stronger signal to be seen. Sera to be tested are added to these wells and
incubated at 37°C, and then washed. If antibodies are present, the antigen-antibody reaction
occurs. No antigen is left for the enzyme-labeled specific HIV antibodies. These antibodies
remain free upon addition and are washed off during washing. Substrate is added, but there
is no enzyme to act on it, so a positive result shows no color change.
5. 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 remaining solution
is passed through a detector for analysis. It has the following steps:
1. Incubation of unlabeled antibody with the sample (antigen): Unlabeled antibody is
incubated in the presence of its antigen (sample). A sufficient incubation period is
provided to allow the antibodies to bind to the antigens.
2. 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.
3. 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.

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4. 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.
Advantages of Reverse ELISA: This test 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 of Reverse ELISA: This test is done, generally, one test at a time and cannot be
done with the micro-titer plate.
ENZYME LINKED IMMUNOSORBENT ASSAY
ADVANTAGES DISADVANTAGES
 Reagents are relatively cheap.
 Reagents have a long shelf life.
 ELISA is highly specific and sensitive.
 No radiation hazards occur during the
labeling or disposal of waste.
 Easy to perform.
 Procedures are quick (not very time
consuming).
 Home test kits, i.e. pregnancy kits, are
inexpensive and widely available.
 ELISA can be used to detect a variety of
infections.
 Measurement of enzyme activity can be
more complex than the measurement of
the 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.
Bottom Line
ELISA tests have their drawbacks and flaws that may overshadow their merits at some points
(as has been elucidated in the table above) 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.).