Enzyme-Linked
Immunosorbent Assay
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Article Contents
• Introduction
• Outline of Methods
Staffan Paulie, Stockholm University, Stockholm, Sweden
Hedvig Perlmann, Stockholm University, Stockholm, Sweden
• Types of ELISA
• Future Developments
• Summary
Based in part on the previous versions of this eLS article ‘Enzyme-linked
Immunosorbent Assay’ (2001, 2006).
Enzyme-linked immunosorbent assay (ELISA) is a
highly sensitive immunoassay utilising enzymeconjugated antibodies, with antigen or antibodies
bound to a solid support. The assay measures
changes in enzyme activities proportional to
the antigen or antibody concentrations involved
in the underlying immune reactions. The assay
exists in many variants and can be designed both
for detection of antigen-specific antibodies and
for quantitative measurements of virtually any
substance. Major applications include diagnostic assessment of disease-related antibodies and
detection and quantitation of hormones, cytokines
and antigens from common pathogens. A special
variant of the assay, ELISpot, can be used for
studying secretion of analytes by single cells and
is widely used for studying immune responses to
vaccination and natural infection at the level of
individual T and B cells.
Introduction
ELISA (enzyme-linked immunosorbent assay; Engvall and
Perlmann, 1971) is a highly versatile and sensitive analytical
test for qualitative or quantitative determination of antibodies or
virtually any kind of antigenically active molecule. Paired with
a simple procedure, it has become one of the most widely used
immunological assays and can be applied for the analysis of
single samples as well as in high-throughput screening. ELISA
may vary in format but always involves the specific interaction
between antibody and antigen, and with one of the reactants
immobilised to a solid support. Immobilisation of an antigen
eLS subject area: Immunology
How to cite:
Paulie, Staffan and Perlmann, Hedvig (January 2016)
Enzyme-Linked Immunosorbent Assay. In: eLS. John Wiley &
Sons, Ltd: Chichester.
DOI: 10.1002/9780470015902.a0002625.pub3
Online posting date: 15th January 2016
makes it possible to measure the binding of specific antibodies
and, correspondingly, attachment of antibodies to the solid
phase allows the detection and quantitation of the antigen. The
antigen–antibody interactions are amplified and visualised by
using enzyme-conjugated reagents, which, depending on the type
of assay used, may be an enzyme-linked anti-immunoglobulin
antibody or a secondary antibody to the specifically bound
antigen. Finally, a chromogenic substrate that gives rise to a
colour change proportional to the intensity of the underlying
immune reaction (Porstmann and Kiessig, 1992) is used. Quantitation is usually obtained by comparing standard samples with
a known concentration of the analyte. Provided antibodies of
high specificity and affinity are available, the detection limits of
the assay is usually well below 1 ng mL−1 . See also: Epitopes;
Immunological Discrimination between Self and Nonself
Outline of Methods
Solid-phase immobilisation of antigens or antibodies is achieved
by linking them, either through adsorption or covalently, to a
suitable matrix (Butler et al., 1992). Such immobilisation makes
use of the capacity of various plastics (e.g. polyvinylchloride or
polystyrene) to adsorb proteins without significantly altering their
immunological properties. The reactants are usually adsorbed on
to the wells of a 96- or 384-well microtitre plate of polystyrene, an
adsorption characterised by strong hydrophobic binding and slow
dissociation rates. After coating with antigen or antibody, the
residual protein-binding capacity of the solid matrix is blocked
by exposing it to an excess of unrelated protein, such as gelatin
or bovine serum albumin. Although the solid support is typically a
microtitre plate, several other surfaces with the appropriate binding characteristics can be used and, for analyses of cellular antigens, living or fixed cells may provide the solid phase. Coating is
followed by the addition of a test solution. For antibody assays,
this may be a serum with an unknown concentration of antibodies
against the immobilised antigen. After incubation and washing,
binding of specific antibodies is revealed and visualised by the
addition of an anti-immunoglobulin–enzyme conjugate followed
by a substrate generating a coloured product when hydrolysed.
The resulting change in colour may be recorded visually or spectrophotometrically and the signal is proportional to the amount
of antibodies bound. Antigens can be measured in a similar way
using competitive- or sandwich-type assays as described below.
See also: Radioactive Labelling of Antibodies
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Enzyme-Linked Immunosorbent Assay
Of the many different enzymes suitable for ELISA, alkaline
phosphatase, horseradish peroxidase and β-galactosidase are the
most commonly used. A wide range of enzyme conjugates with
different specificities are commercially available, but specific
conjugates may also be prepared by mixing and cross-linking
antibodies with the desired specificity to the enzyme using glutaraldehyde or more specific cross-linking procedures. As an
alternative to using enzyme-conjugated antibodies or antigens,
a versatile amplification system involves antibodies or antigens conjugated with biotin, a low-molecular-weight member
of the vitamin B complex. The biotinylated antibodies or antigens can then be combined with a general reagent comprised
of enzyme-conjugated streptavidin, a bacterial protein that binds
biotin with high affinity (Dundas et al., 2013).
Types of ELISA
Indirect ELISA to determine specific
antibodies
The indirect ELISA, designed to measure antibody binding to
immobilised antigen, is particularly suitable for screening of
antigen-specific antibodies in serum, plasma or other biological
fluids. It is the prototype of many serologically based diagnostic assays, where the immobilised antigens typically are antigenic components from bacteria or viruses, allergens or target
antigens for autoimmune reactions. After antibody binding and
the removal of unbound antibodies, detection is achieved by a
secondary enzyme-conjugated anti-immunoglobulin reagent followed by a chromogenic substrate. The amount of coloured product is quantitated spectrophotometrically in an ELISA reader, and
the results may be scored either as positive or negative in relation
to a certain cut-off level. If required, the antibody concentration
in the test serum may be more precisely determined by comparison with a standard curve, constructed using different dilutions
of a solution containing known concentrations of immunoglobulin of the same animal origin as the antibodies in the test serum
(Berzofsky et al., 1999). Because of the molecular heterogeneity of the immunoglobulins, antibody concentrations are usually
expressed as units of antibody activity per volume rather than as
weight units. For quantitation, it is necessary to use optimal concentrations of all reagents, that is, the solid antigen should be in
excess so that the number of antibodies in the test serum becomes
the limiting factor.
Due to the high content of immunoglobulin, serum or plasma
samples often need to be diluted (e.g. 50 to 100 times) before
being tested to avoid background and false positives caused by
antibodies binding to the microplate wells in a non-specific manner. As dilution will reduce sensitivity, a modified version of the
assay has been developed where the anti-immunoglobulin detection reagent is exchanged for a biotin- or enzyme-conjugated
variant of the same antigen as used for coating the plates.
Often referred to as the double-antigen-sandwich ELISA or
antigen-bridging ELISA, this assay format is not affected by
the presence of non-specifically bound immunoglobulin. With no
need for sample dilution and with an effective detection of all Ig
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classes including IgM, normally the first antibody to be induced
during an immune response, the assay can provide an earlier diagnostic answer after an infection (Hu et al., 2008). The format is
also often used to monitor the immunogenicity of protein drugs
by looking for the induction of anti-drug antibodies (Liu et al.,
2011). See also: Autoimmune Disease: Diagnosis
Direct competitive ELISA to determine
soluble antigen
This assay is useful both for quantitating soluble antigens and for
studying antigenic specificities of any molecules for which antibodies are available. Microtitre plates are coated with a known
antigen and a fixed concentration of antibody is added in the
presence or absence of the same antigen in solution. If antigen
is present in an unknown sample, part of the antibodies will be
blocked from binding to the solid-phase antigen, resulting in a
decreased signal. The amount of antigen in the test solution is
proportional to the inhibition of antibody binding, and quantitation is achieved by comparing with a standard curve obtained
with serial dilutions of an antigen solution of known concentration (Paulie et al., 2006). The assay is particularly useful for the
analysis of smaller molecules, which, due to epitope restriction,
cannot be analysed by the more sensitive sandwich ELISA (see
below).
Antibody sandwich ELISA to determine
soluble antigen
Similar to the competitive ELISA, sandwich or capture ELISA
can be used to quantify virtually any soluble molecule as long
as it is large enough to harbour two separate antigenic sites (epiotpes) to which antibodies can be bound simultaneously without
interference. Although both polyclonal and monoclonal reagents
can be used, the assay typically involves two monoclonal antibodies, one serving as an immobilised capture antibody and the
other directed at a distinct epitope, serving as a detection antibody
(Figure 1).
In the first step, the wells of microtitre plates are coated with
capture antibody specific for the test antigen. A sample containing
an unknown amount of the antigen is added, and is followed by
the incubation with a biotinylated (or enzyme-conjugated) detection antibody recognising a different epitope than the capture antibody. Finally, the wells are incubated with enzyme-conjugated
streptavidin, followed by substrate. The concentration of antigen in the test solution is proportional to the amount of substrate
hydrolysed and may be determined against a known standard, as
indicated above (Paulie et al., 2006).
The sandwich ELISA is significantly more sensitive than the
competitive ELISA and, because it requires the detection of
two separate epitopes, cross-reactivity with structurally related
molecules is rare. However, the assay is critically dependent on
the quality and properties of the capture and detection antibodies
in order to be robust and optimally sensitive.
Commercial assays based on this technique, and containing
precoated plates and simplified procedures, are available for a
large number of antigens.
eLS © 2016, John Wiley & Sons, Ltd. www.els.net
Enzyme-Linked Immunosorbent Assay
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The assay is commonly performed in 96-well
microtitre plates. In the first step the wells are
coated with a monoclonal antibody (Y) specific
for the antigen to be determined
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Samples containing unknown amounts of the
antigen ( ) are added to the wells. To other wells,
a standard of known concentration is added.
During the incubation, the antigen is captured by
the antibody attacthed to the wells
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The sample/standard is removed by washing and
a biotinylated antibody ( ) is added
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Streptavidin-enzyme conjugate ( ) is added
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Figure 1
Finally, a chromogenic substrate for the enzyme is
added and the plates are developed until colour
emerges. Within the detection range of the assay,
the intensity of the colour is directly proportional
to the amount of antigen added to each well. The
concentration of samples is determined by
comparison to the standard
Schematic outline of a sandwich or capture ELISA.
A potential and often unrecognised problem with the sandwich ELISA as well as other sandwich-based assays, when
employed for testing serum or plasma, is the interference by heterophilic antibodies. These antibodies, sometimes also referred
to as HAMA (human anti-mouse antibodies), are present in the
blood of many people and are reactive with immunoglobulins
from other species. By binding to the immobilised capture antibody with one ‘arm’ and to the detection antibody with the other,
heterophilic antibodies may generate a false-positive signal that
is indistinguishable from that seen with antigen (Sehlin et al.,
2010). Commercially available ELISA kits designed for testing
serum or plasma usually include an incubation buffer with free
antibody which, in a competitive manner, prevents heterophilic
antibodies from interacting with the assay antibodies.
ELISpot assay to enumerate cells
secreting cytokines, antibodies or other
soluble factors
The ELISpot (enzyme-linked immunospot) assay is technically
similar to the ELISA; but rather than measuring a substance in
solution, it detects and measures the number of cells secreting the
same substance. While originally developed for the enumeration
of B cells producing antigen-specific antibodies, the technique is
today primarily used to analyse specific T-cell responses. This
is achieved by measuring the secretion of cytokine by individual T cells after activation with antigen (Czerkinsky et al., 1988;
Lalvani et al., 1997). As in the sandwich ELISA, a capture antibody is coated onto a solid support, usually a 96-well microtitre
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Enzyme-Linked Immunosorbent Assay
Future Developments
(a)
(b)
Figure 2 ELISpot showing T cells responding with Interferon-γ secretion
after 18 h of exposure in vitro to antigenic peptides from cytomegalovirus,
CMV (a). Each spot indicates a responding cell and a control well (b) that
without addition of antigen shows no or few secreting cells.
plate with a filter membrane bottom. The membrane, preferably
of polyvinylidene difluoride (PVDF) or nitrocellulose is characterised by a high capacity to adsorb protein (50–100 times that of
an ELISA plate), a prerequisite to obtain a sufficiently high concentration of antibody to allow most of the cytokine to be captured
at the site of the secreting cell. The cells are then added together
with stimuli, normally a specific antigen or polyclonal activator, and incubated under sterile conditions, allowing cell activation and local binding of the secreted product on the membrane.
After removing cells by washing, enzyme-conjugated antibody
(or alternatively biotinylated antibody plus enzyme-conjugated
streptavidin) is added. Finally, incubation with a precipitating
substrate results in the formation of spots, each corresponding
to an activated T cell secreting cytokine (Figure 2). The number
of activated cells can be determined by counting the spots in a
dissection microscope or in an ELISpot reader, which may also
provide information about spot size and intensity.
With a detection level that can be as low as 1 cell in 100 000, the
ELISpot is between 20 and 200 times more sensitive than ELISA.
Due to its high sensitivity, it has proven particularly useful when
studying small populations of active cells, such as those regularly found in specific immune responses, and it has found wide
applications as a tool in analysing natural T-cell responses as well
as those induced by vaccination (Novitsky et al., 2001; Wang
et al., 2001; Slota et al., 2011). The method has also recently been
exploited for diagnostic purposes with the best example being the
T-Spot. TB assay for diagnosis of tuberculosis infection. This
assay, which measures the number of Interferon-γ-secreting T
cells after challenge with selected antigens from Mycobacterium
tuberculosis, has been shown to be more sensitive and accurate than the tuberculin skin test (Whitworth et al., 2013). By
analysing different cytokines, the ELISpot can also provide qualitative information about the type of responding T cells as different
cells, for example, cytotoxic T cells and helper T cells, are characterised by the release of different cytokines when activated. Apart
from T cells, the method has also been used to analyse cytokine
secretion by other immune cells including monocytes and dendritic cells (Smedman et al., 2009) and is also increasingly being
used for analysis of antibody responses by individual B cells, the
purpose for which the technique was originally described. See
also: T Lymphocytes: Helpers
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ELISA technology, which has already been fully automated, can
today be used to assay practically any kind of substance to which
antibodies can be produced. Development of new antibodies not
only for human antigens but also for the veterinary field continuously extends the number of available assays. Higher sensitivities
and more rapid assays may be achieved by using fluorogenic
instead of chromogenic substrates and through amplification systems comprising fluorochromes or chemiluminescence. The use
of fluorescently labelled detection reagents has also enabled
simultaneous analysis of multiple antigens or antibody reactivities. The solid phase may here be a microchip or mixtures of
differently labelled microbeads where each analyte is separately
measured at different locations of the chip or on beads with a
unique signature. Micoarray or multiplex assays of this type are
commercially available and are often provided as kits comprising
a number of analytes linked to a specific field of research. A similar development is being seen for the ELISpot where the use of
fluorophore-labelled detection reagents in the FluoroSpot assay
allows analysis of multiple analytes at the level of single cells.
Furthermore, the possibility of producing antibody fragments
and other affinity-binding molecules via phage display and other
technologies will help extend the number of substances that are
possible to analyse. These approaches are particularly valuable
in situations where generation of conventional antibodies may be
difficult due to functional properties of the antigen (e.g. cytotoxic
or immunosuppressive) or when the antigen is poorly immunogenic. Finally, the ELLISA format can also be used to measure
other specific molecular interactions, for example, between hormones or cytokines and their receptors (Vieira, 1998). See also:
Cytokine Assays; Autoimmune Disease: Diagnosis
Summary
ELISA is a versatile and sensitive analytical technique for the
qualitative or quantitative determination of both antibodies and
any kind of antigenic substance. With either antigen or antibody
bound to a solid support, it allows binding of the corresponding
reactant in a test solution and subsequent separation of unbound
reactant by a simple washing procedure. The antigen–antibody
interactions are revealed by the use of an enzyme-conjugated
detecting reagent, together with substrates that generate a
coloured reaction product proportional to the strength of the
immune reaction. ELISA exists in a great number of variants
suitable for quantitating antibodies or antigens. A special variant,
the ELISpot assay, is being employed to enumerate secretory
cells, for example, T cells secreting cytokines in response
to antigenic challenge or B cells producing antigen-specific
antibodies.
References
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eLS © 2016, John Wiley & Sons, Ltd. www.els.net
Enzyme-Linked Immunosorbent Assay
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Further Reading
Butler JE (1994) Enzyme linked immunosorbent assay. In: van
Oss CJ and van Regenmortel MHV (eds) Immunochemistry,
pp. 759–803. New York: Marcel Dekker.
Calarota SA and Baldanti F (2013) Enumeration and characterization of human memory T cells by enzyme-linked immunospot
assays. Clinical and Developmental Immunology, 2013, Article ID
637649.
Hornbeck P (1991) Enzyme-linked immunosorbent assay. In: Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM and Strober
W (eds) Current Protocols in Immunology, pp. 2.1.1–2.1.22. New
York: Greene Publishing Associates & Wiley Interscience.
Klinman DM and Nutman TB (1994) ELISPOT assay to detect
cytokine secreting murine and human cells. In: Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM and Strober W (eds)
Current Protocols in Immunology, pp. 6.19.1–6.19.8. New York:
Greene Publishing Associates & Wiley.
Nielsen UB and Geierstanger BH (2004) Multiplexed sandwich
assays in microarray format. Journal of Immunological Methods
290: 107–120.
Stott DI (1994) Immunoblotting, dot-blotting, and ELISPOT assays:
methods and application. In: van Oss CJ and van Regenmortel
MHV (eds) Immunochemistry, pp. 925–948. New York: Marcel
Dekker.
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