The enzyme-linked immunosorbent assay is a test that uses antibodies and color change
to identify a substance. ELISA is a popular format of "wet-lab" type
analytic biochemistry assay that uses a solid-phase enzyme immunoassay to detect the presence
of a substance, usually an antigen, in a liquid sample or wet sample.
The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as
a quality-control check in various industries. Antigens from the sample are attached to a
surface. Then, a further specific antibody is applied over the surface so it can bind
to the antigen. This antibody is linked to an enzyme, and, in the final step, a substance
containing the enzyme's substrate is added. The subsequent reaction produces a detectable
signal, most commonly a color change in the substrate.
Performing an ELISA involves at least one antibody with specificity for a particular
antigen. The sample with an unknown amount of antigen is immobilized on a solid support
either non-specifically or specifically. After the antigen is immobilized, the detection
antibody is added, forming a complex with the antigen. The detection antibody can be
covalently linked to an enzyme, or can itself be detected by a secondary antibody that is
linked to an enzyme through bioconjugation. Between each step, the plate is typically
washed with a mild detergent solution to remove any proteins or antibodies that are aspecifically
bound. After the final wash step, the plate is developed by adding an enzymatic substrate
to produce a visible signal, which indicates the quantity of antigen in the sample.
Of note, ELISA can perform other forms of ligand binding assays instead of strictly
"immuno" assays, though the name carried the original "immuno" because of the common use
and history of development of this method. The technique essentially requires any ligating
reagent that can be immobilized on the solid phase along with a detection reagent that
will bind specifically and use an enzyme to generate a signal that can be properly quantified.
In between the washes, only the ligand and its specific binding counterparts remain specifically
bound or "immunosorbed" by antigen-antibody interactions to the solid phase, while the
nonspecific or unbound components are washed away. Unlike other spectrophotometric wet
lab assay formats where the same reaction well can be reused after washing, the ELISA
plates have the reaction products immunosorbed on the solid phase which is part of the plate,
so are not easily reusable.
Principle As an analytic biochemistry assay, ELISA involves
detection of an "analyte" in a liquid sample by a method that continues to use liquid reagents
during the "analysis" that stays liquid and remains inside a reaction chamber or well
needed to keep the reactants contained; It is opposed to "dry lab" that can use dry strips
- and even if the sample is liquid, the final detection step in "dry" analysis involves
reading of a dried strip by methods such as reflectometry and does not need a reaction
containment chamber to prevent spillover or mixing between samples.
As a heterogenous assay, ELISA separates some component of the analytical reaction mixture
by adsorbing certain components onto a solid phase which is physically immobilized. In
ELISA, a liquid sample is added onto a stationary solid phase with special binding properties
and is followed by multiple liquid reagents that are sequentially added, incubated and
washed followed by some optical change in the final liquid in the well from which the
quantity of the analyte is measured. The qualitative "reading" usually based on detection of intensity
of transmitted light by spectrophotometry, which involves quantitation of transmission
of some specific wavelength of light through the liquid. The sensitivity of detection depends
on amplification of the signal during the analytic reactions. Since enzyme reactions
are very well known amplification processes, the signal is generated by enzymes which are
linked to the detection reagents in fixed proportions to allow accurate quantification
- thus the name "enzyme linked". The analyte is also called the ligand because
it will specifically bind or ligate to a detection reagent, thus ELISA falls under the bigger
category of ligand binding assays. The ligand-specific binding reagent is "immobilized", i.e., usually
coated and dried onto the transparent bottom and sometimes also side wall of a well, which
is usually constructed as a multiple-well plate known as the "ELISA plate". Conventionally,
like other forms of immunoassays, the specificity of antigen-antibody type reaction is used
because it is easy to raise an antibody specifically against an antigen in bulk as a reagent. Alternatively,
if the analyte itself is an antibody, its target antigen can be used as the binding
reagent. History
Before the development of the ELISA, the only option for conducting an immunoassay was radioimmunoassay,
a technique using radioactively labeled antigens or antibodies. In radioimmunoassay, the radioactivity
provides the signal, which indicates whether a specific antigen or antibody is present
in the sample. Radioimmunoassay was first described in a scientific paper by Rosalyn
Sussman Yalow and Solomon Berson published in 1960.
Because radioactivity poses a potential health threat, a safer alternative was sought. A
suitable alternative to radioimmunoassay would substitute a nonradioactive signal in place
of the radioactive signal. When enzymes react with appropriate substrates, a change in color
occurs, which is used as a signal. However, the signal has to be associated with the presence
of antibody or antigen, which is why the enzyme has to be linked to an appropriate antibody.
This linking process was independently developed by Stratis Avrameas and G. B. Pierce. Since
it is necessary to remove any unbound antibody or antigen by washing, the antibody or antigen
has to be fixed to the surface of the container; i.e., the immunosorbent must be prepared.
A technique to accomplish this was published by 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. Traditional ELISA typically involves chromogenic
reporters and substrates that produce some kind of observable color change to indicate
the presence of antigen or analyte. Newer ELISA-like techniques use fluorogenic, electrochemiluminescent,
and quantitative PCR reporters to create quantifiable signals. These new reporters can have various
advantages, including higher sensitivities and multiplexing. In technical terms, newer
assays of this type are not strictly ELISAs, as they are not "enzyme-linked", but are instead
linked to some nonenzymatic reporter. However, given that the general principles in these
assays are largely similar, they are often grouped in the same category as ELISAs.
In 2012 an ultrasensitive, enzyme-based ELISA test using nanoparticles as a chromogenic
reporter was able to give a naked-eye colour signal from the detection of mere attograms
of analyte. A blue color appears for positive results and red color for negative. Note that
this detection only can confirm the presence or the absence of analyte not the actual concentration.
Types "Indirect" ELISA
The steps of "Indirect" ELISA follows the mechanism below:-
A buffered solution of the antigen to be tested for is added to each well of a microtiter
plate, where it is given time to adhere to the plastic through charge interactions.
A solution of nonreacting protein, such as bovine serum albumin or casein, is added to
well any plastic surface in the well that remains uncoated by the antigen.
The primary antibody is added, which binds specifically to the test antigen coating the
well. This primary antibody could also be in the serum of a donor to be tested for reactivity
towards the antigen. A secondary antibody is added, which will
bind the primary antibody. This secondary antibody often has an enzyme attached to it,
which has a negligible effect on the binding properties of the antibody. In other cases,
as in the diagram to the left, the primary antibody itself is conjugated to the enzyme.
A substrate for this enzyme is then added. Often, this substrate changes color upon reaction
with the enzyme. The color change shows the secondary antibody has bound to primary antibody,
which strongly implies the donor has had an immune reaction to the test antigen. This
can be helpful in a clinical setting, and in research.
The higher the concentration of the primary antibody present in the serum, the stronger
the color change. Often, a spectrometer is used to give quantitative values for color
strength. The enzyme acts as an amplifier; even if only
few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal
molecules. Within common-sense limitations, the enzyme can go on producing color indefinitely,
but the more primary antibody is present in the donor serum, the more secondary antibody
+ enzyme will bind, and the faster the color will develop. A major disadvantage of the
indirect 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 well,
so small concentrations of analyte in serum must compete with other serum proteins when
binding to the well surface. The sandwich or direct ELISA provides a solution to this
problem, by using a "capture" antibody specific for the test antigen to pull it out of the
serum's molecular mixture. ELISA may be run in a qualitative or quantitative
format. Qualitative results provide a simple positive or negative result for a sample.
The cutoff between positive and negative is determined by the analyst and may be statistical.
Two or three times the standard deviation is often used to distinguish positive from
negative samples. In quantitative ELISA, the optical density of the sample is compared
to a standard curve, which is typically a serial dilution of a known-concentration solution
of the target molecule. For example, if a test sample returns an OD of 1.0, the point
on the standard curve that gave OD = 1.0 must be of the same analyte concentration as the
sample. Sandwich ELISA
A "sandwich" ELISA, is used to detect sample antigen. The steps are:
A surface is prepared to which a known quantity of capture antibody is bound.
Any nonspecific binding sites on the surface are blocked.
The antigen-containing sample is applied to the plate.
The plate is washed to remove unbound antigen. A specific antibody is added, and binds to
antigen Enzyme-linked secondary antibodies are applied
as detection antibodies that also bind specifically to the antibody's Fc region.
The plate is washed to remove the unbound antibody-enzyme conjugates.
A chemical is added to be converted by the enzyme into a color or fluorescent or electrochemical
signal. The absorbency or fluorescence or electrochemical
signal of the plate wells is measured to determine the presence and quantity of antigen.
The image to the right includes the use of a secondary antibody conjugated to an enzyme,
though, in the technical sense, this is not necessary if the primary antibody is conjugated
to an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process of
creating enzyme-linked antibodies for every antigen one might want to detect. By using
an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked
antibody can be used in a variety of situations. Without the first layer of "capture" antibody,
any proteins in the sample 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. A descriptive animation of the application
of sandwich ELISA to home pregnancy testing can be found here.
Competitive ELISA A third use of ELISA is through competitive
binding. The steps for this ELISA are somewhat different from the first two examples:
Unlabeled antibody is incubated in the presence of its antigen.
These bound antibody/antigen complexes are then added to an antigen-coated well.
The plate is washed, so unbound antibody is removed.
The secondary antibody, specific to the primary antibody, is added. This second antibody is
coupled to the enzyme. A substrate is added, and remaining enzymes
elicit a chromogenic or fluorescent signal. The reaction is stopped to prevent eventual
saturation of the signal. 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. 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.
For the detection of HIV antibodies, the wells of microtiter plate are coated with the HIV
antigen. Two specific antibodies are used, one conjugated with enzyme and the other present
in serum. 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-labelled 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 positive result shows no color change.
Animated video overview of competitive ELISA. Multiple and ready to use ELISA
A new technique uses a solid phase made up of an immunosorbent polystyrene rod with eight
to 12 protruding ogives. The entire device is immersed in a test tube containing the
collected sample and the following steps are carried out by dipping the ogives in microwells
of standard microplates filled with reagents. The advantages of this technique are:
The ogives can each be sensitized to a different reagent, allowing the simultaneous detection
of different antibodies and/or different antigens for multiple-target assays.
The sample volume can be increased to improve the test sensitivity in clinical, food and
environmental samples. One ogive is left unsensitized to measure
the nonspecific reactions of the sample. The use of laboratory supplies for dispensing
sample aliquots, washing solution and reagents in microwells is not required, facilitating
the development of ready-to-use lab kits and on-site testing.
Applications
Because the ELISA can be performed to evaluate either the presence of antigen or the presence
of antibody in a sample, it is a useful tool for determining serum antibody concentrations.
It has also found applications in the food industry in detecting potential food allergens,
such as milk, peanuts, walnuts, almonds, and eggs. ELISA can also be used in toxicology
as a rapid presumptive screen for certain classes of drugs.
The ELISA was the first screening test widely used for HIV because of its high sensitivity.
In an ELISA, a person's serum is diluted 400 times and applied to a plate to which HIV
antigens are attached. If antibodies to HIV are present in the serum, they may bind to
these HIV antigens. The plate is then washed to remove all other components of the serum.
A specially prepared "secondary antibody" — an antibody that binds to other antibodies
— is then applied to the plate, followed by another wash. This secondary antibody is
chemically linked in advance to an enzyme. Thus, the plate will contain enzyme in proportion
to the amount of secondary antibody bound to the plate. A substrate for the enzyme is
applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA
results are reported as a number; the most controversial aspect of this test is determining
the "cut-off" point between a positive and a negative result.
A cut-off point may be determined by comparing it with a known standard. If an ELISA test
is used for drug screening at workplace, a cut-off concentration, 50 ng/ml, for example,
is established, and a sample containing the standard concentration of analyte will be
prepared. Unknowns that generate a stronger signal than the known sample are "positive."
Those that generate weaker signal are "negative". Dr Dennis E Bidwell and Alister Voller created
the ELISA test to detect various kind of diseases, such as malaria, Chagas disease, and Johne's
disease. ELISA tests also are used as in in vitro diagnostics in medical laboratories.
The other uses of ELISA include: detection of Mycobacterium antibodies in tuberculosis
detection of rotavirus in feces detection of hepatitis B markers in serum
detection of enterotoxin of E. coli in feces detection of HIV antibodies in blood samples
See also Immunoscreening
Lateral flow test Magnetic immunoassay
Plaque reduction neutralization test Secretion assay
Notes and references
External links