ELISA

For other uses, see ELISA (disambiguation).

The enzyme-linked immunosorbent assay (ELISA) (/laz/, /ilaz/) 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 solidphase enzyme immunoassay (EIA) 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
(usually a polystyrene microtiter plate) either non-specifically (via adsorption to the
surface) or specifically (via capture by another antibody specific to the same antigen, in a
"sandwich" ELISA). 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 non-specifically 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 (e.g. a cuvette)
can be reused after washing, the ELISA plates have the reaction products immunosorbed
on the solid phase which is part of the plate, and so are not easily reusable.

Principle
As an analytic biochemistry assay, ELISA involves detection of an "analyte" (i.e. the
specific substance whose presence is being quantitatively or qualitatively analyzed) in a
liquid sample by a method that continues to use liquid reagents during the "analysis" (i.e.
controlled sequence of biochemical reactions that will generate a signal which can be
easily quantified and interpreted as a measure of the amount of analyte in the sample)
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 (e.g. a measured small drop), 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 (e.g. color development by the
product of an enzymatic reaction) 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 (as well as the transparent bottom of
the well in the multiple-well plate format). 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 ligandspecific binding reagent is "immobilized", i.e., usually coated and dried onto the
transparent bottom and sometimes also side wall of a well (the stationary "solid
phase'/"solid substrate" here as opposed to solid microparticle/beads that can be washed
away), 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 (such as horseradish peroxidase) react with
appropriate substrates (such as ABTS or TMB), 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
Direct ELISA
The steps of direct 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 (usually 96-well plates)
in order to cover any plastic surface in the well which
remains uncoated by the antigen.
The primary antibody with an attached (conjugated)
enzyme is added, which binds specifically to the test
antigen coating the well.
A substrate for this enzyme is then added. Often, this
substrate changes color upon reaction with the enzyme.
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 enzymelinked 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 antibody is bound, the faster the color will develop. 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 well, so small concentrations of analyte in serum must compete with
other serum proteins when binding to the well surface. The sandwich or indirect 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 (yes or no) 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 (error inherent in a test) is often used to distinguish positive from
negative samples. In quantitative ELISA, the optical density (OD) of the sample is
compared to a standard curve, which is typically a serial dilution of a knownconcentration 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.
The use and meaning of the names "direct ELISA" and "indirect ELISA" differs in the
literature and on web sites depending on the context of the experiment. When the
presence of an antigen is analyzed, the name "direct ELISA" refers to an ELISA in which
only a labelled primary antibody is used, and the term "indirect ELISA" refers to an ELISA
in which the antigen is bound by the primary antibody which then is detected by a labeled
secondary antibody. In the latter case a sandwich ELISA is clearly distinct from an indirect
ELISA. When the "primary" antibody is of interest, e.g. in the case of immunization
analyses, this antibody is directly detected by the secondary antibody and the term
"direct ELISA" applies to a setting with two antibodies.

Sandwich ELISA
A "sandwich" ELISA is used to detect sample antigen. The steps are:
1. A surface is prepared to which a known quantity of capture antibody is bound.
2. Any nonspecific binding sites on the surface are blocked.
3. The antigen-containing sample is applied to the plate, and captured by antibody.
4. The plate is washed to remove unbound antigen.
5. A specific antibody is added, and binds to antigen (hence the 'sandwich': the Ag is
stuck between two antibodies). This primary antibody could also be in the serum of
a donor to be tested for reactivity towards the antigen.
6. Enzyme-linked secondary antibodies are applied as detection antibodies that also
bind specifically to the antibody's Fc region (nonspecific).
7. The plate is washed to remove the unbound antibody-enzyme conjugates.
8. A chemical is added to be converted by the enzyme into a color or fluorescent or
electrochemical signal.
9. The absorbance or fluorescence or electrochemical signal (e.g., current) 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 (which would be direct ELISA). However, the 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 (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. A sandwich ELISA used for research often
need validation because of the risk of false positive results.

Competitive ELISA
A third use of ELISA is through competitive binding. The steps for this ELISA are
somewhat different from the first two examples:
1. Unlabeled antibody is incubated in the presence of its antigen (sample).
2. These bound antibody/antigen complexes are then added to an antigen-coated well.
3. The plate is washed, so unbound antibodies are removed. (The more antigen in the
sample, the more Ag-Ab complexes are formed and so there are less unbound
antibodies available to bind to the antigen in the well, hence "competition".)
4. The secondary antibody, specific to the primary antibody, is added. This second
antibody is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent
signal.
6. 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 (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.
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 (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-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
a positive result shows no color change.

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 (such as with the HIV test or West Nile virus). It has also found
applications in the food industry in detecting potential food allergens, such as milk,
peanuts, walnuts, almonds, and eggs and as serological blood test for coeliac disease.
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
microtitre plate
Plaque reduction neutralization test
Plate reader
Secretion assay
Agglutination-PCR

Notes and references

1. ^ Yalow, Rosalyn S.; Berson, Solomon A. (1960). "Immunoassay of endogenous
plasma insulin in man". The Journal of Clinical Investigation 39: 115775.

doi:10.1172/JCI104130. PMC 441860. PMID 13846364.

2. ^ Lequin, R. M. (2005). "Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent
Assay (ELISA)". Clinical Chemistry 51 (12): 24158.
doi:10.1373/clinchem.2005.051532. PMID 16179424.
3. ^ Wide, Leif; Porath, Jerker (1966). "Radioimmunoassay of proteins with the use of

Sephadex-coupled antibodies". Biochimica et Biophysica Acta 130 (1): 25760.

doi:10.1016/0304-4165(66)90032-8.
4. ^ Engvall, Eva; Perlmann, Peter (1971). "Enzyme-linked immunosorbent assay
(ELISA) quantitative assay of immunoglobulin G". Immunochemistry 8 (9): 8714.
doi:10.1016/0019-2791(71)90454-X. PMID 5135623.

5. ^ Van Weemen, B.K.; Schuurs, A.H.W.M. (1971). "Immunoassay using antigen

enzyme conjugates". FEBS Letters 15 (3): 232236. doi:10.1016/00145793(71)80319-8. PMID 11945853.
6. ^ Leng, S. X.; McElhaney, J. E.; Walston, J. D.; Xie, D.; Fedarko, N. S.; Kuchel, G. A.
(2008). "ELISA and Multiplex Technologies for Cytokine Measurement in
Inflammation and Aging Research". The Journals of Gerontology Series A: Biological
Sciences and Medical Sciences 63 (8): 87984. doi:10.1093/gerona/63.8.879.
PMC 2562869. PMID 18772478.

7. ^ Adler, Michael; Schulz, Sven; Spengler, Mark (2009). "Cytokine Quantification in

Drug Development: A comparison of sensitive immunoassay platforms". Chimera
Biotech.
8. ^ de la Rica, Roberto; Stevens, Molly M. (2012). "Plasmonic ELISA for the
ultrasensitive detection of disease biomarkers with the naked eye". Nature

Nanotechnology 7 (12): 8214. doi:10.1038/nnano.2012.186. PMID 23103935.

9. ^ Schmidt, SD; Mazzella, MJ; Nixon, RA; Mathews, PM (2012). "A! measurement by
enzyme-linked immunosorbent assay". Methods in Molecular Biology 849: 50727.
doi:10.1007/978-1-61779-551-0_34. PMID 22528112.
10. ^ Kragstrup, Tue W; Vorup-Jensen, Thomas; Deleuran, Bent; Hvid, Malene (2013). "A
simple set of validation steps identifies and removes false results in a sandwich
enzyme-linked immunosorbent assay caused by anti-animal IgG antibodies in
plasma from arthritis patients". SpringerPlus 2 (1): 263. doi:10.1186/2193-1801-2263.
11. ^ MedlinePlus Encyclopedia ELISA/Western blot tests for HIV

12. ^ "Food Allergen Partnership" (Press release). FDA. January 2001. Retrieved August
20, 2015.
13. ^ Sblattero, D.; Berti, I.; Trevisiol, C.; Marzari, R.; Tommasini, A.; Bradbury, A.;
Fasano, A.; Ventura, A.; Not, T. (2000). "Human recombinant tissue
transglutaminase ELISA: an innovative diagnostic assay for celiac disease". The

American Journal of Gastroenterology 95 (5): 12537. doi:10.1111/j.15720241.2000.02018.x. PMID 10811336.

14. ^ Porcelli, Brunetta; Ferretti, Fabio; Vindigni, Carla; Terzuoli, Lucia (2014).
"Assessment of a Test for the Screening and Diagnosis of Celiac Disease". Journal of
Clinical Laboratory Analysis. doi:10.1002/jcla.21816. PMID 25385391.

15. ^ Griffin, J. F. T.; Spittle, E.; Rodgers, C. R.; Liggett, S.; Cooper, M.; Bakker, D.;
Bannantine, J. P. (2005). "Immunoglobulin G1 Enzyme-Linked Immunosorbent Assay
for Diagnosis of Johne's Disease in Red Deer (Cervus elaphus)". Clinical and Vaccine
Immunology 12 (12): 14019. doi:10.1128/CDLI.12.12.1401-1409.2005. PMC 1317074.
PMID 16339063.

External links