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ELISA
From Wikipedia, the free encyclopedia

The enzyme-linked immunosorbent assay (ELISA) (/laz/,

/ilaz/) is a test that uses antibodies and color change to ELISA
identify a substance.

ELISA is a popular format of "wet-lab" type analytic

biochemistry assay that uses a solid-phase 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 A 96-well microtiter plate being used for ELISA
further specific antibody is applied over the surface so it can
bind to the antigen. This antibody is linked to an enzyme, and, in MeSH D004797
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.

Contents
1 Principle
2 History
3 Types
3.1 Direct ELISA
3.2 Sandwich ELISA
3.3 Competitive ELISA
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4 Applications
5 See also
6 Notes and references
7 External links

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 ligand-specific 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.[1]

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.

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by Stratis Avrameas and G. B. Pierce.[2]

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.[3]

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.[4][5]

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
A paramedic assistant prepares analyses
quantifiable signals. These new reporters can have various advantages, in an ELISA laboratory
including higher sensitivities and multiplexing.[6][7] 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.[8]

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 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 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
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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 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.

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 "indirect ELISA" applies to a setting with two
antibodies.

Sandwich ELISA

A "sandwich" ELISA is
used to detect sample
antigen.[9] The steps are:

1. A surface is prepared to
which a known quantity
of capture antibody is A sandwich ELISA. (1) Plate is coated with a capture
Direct ELISA diagram bound. antibody; (2) sample is added, and any antigen present
2. Any nonspecific binding binds to capture antibody; (3) detecting antibody is
sites on the surface are added, and binds to antigen; (4) enzyme-linked
blocked. secondary antibody is added, and binds to detecting
3. The antigen-containing sample is applied to the plate, antibody; (5) substrate is added, and is converted by
and captured by antibody. enzyme to detectable form.
4. The plate is washed to remove unbound antigen.
5. A specific antibody is added, and binds to antigen
(hence the 'sandwich': the antigen 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
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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.[10]

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[11]
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[12] and as serological blood test for coeliac disease.[13][14] ELISA
can also be used in toxicology as a rapid presumptive screen for certain
classes of drugs.
Human anti-IgG, double antibody
The ELISA was the first screening test widely used for HIV because of its
sandwich ELISA
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.

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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. Enzyme-linked immunosorbent assay
Unknowns that generate a stronger signal than the known sample are plate
"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.[15] 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" (http
s://www.ncbi.nlm.nih.gov/pmc/articles/PMC441860). The Journal of Clinical Investigation. 39: 115775.
PMC 441860 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC441860) . PMID 13846364 (https://www.nc
bi.nlm.nih.gov/pubmed/13846364). doi:10.1172/JCI104130 (https://doi.org/10.1172%2FJCI104130).
2. Lequin, R. M. (2005). "Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA)".
Clinical Chemistry. 51 (12): 24158. PMID 16179424 (https://www.ncbi.nlm.nih.gov/pubmed/16179424).
doi:10.1373/clinchem.2005.051532 (https://doi.org/10.1373%2Fclinchem.2005.051532).
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 (https://do
i.org/10.1016%2F0304-4165%2866%2990032-8).
4. Engvall, Eva; Perlmann, Peter (1971). "Enzyme-linked immunosorbent assay (ELISA) quantitative assay of
immunoglobulin G". Immunochemistry. 8 (9): 8714. PMID 5135623 (https://www.ncbi.nlm.nih.gov/pubme
d/5135623). doi:10.1016/0019-2791(71)90454-X (https://doi.org/10.1016%2F0019-2791%2871%2990454-
X).

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5. Van Weemen, B.K.; Schuurs, A.H.W.M. (1971). "Immunoassay using antigenenzyme conjugates". FEBS
Letters. 15 (3): 232236. PMID 11945853 (https://www.ncbi.nlm.nih.gov/pubmed/11945853).
doi:10.1016/0014-5793(71)80319-8 (https://doi.org/10.1016%2F0014-5793%2871%2980319-8).
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" (https://www.ncbi.
nlm.nih.gov/pmc/articles/PMC2562869). The Journals of Gerontology Series A: Biological Sciences and
Medical Sciences. 63 (8): 87984. PMC 2562869
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2562869) . PMID 18772478 (https://www.ncbi.nlm.nih.go
v/pubmed/18772478). doi:10.1093/gerona/63.8.879 (https://doi.org/10.1093%2Fgerona%2F63.8.879).
7. Adler, Michael; Schulz, Sven; Spengler, Mark (2009). "Cytokine Quantification in Drug Development: A
comparison of sensitive immunoassay platforms" (http://www.bionity.com/en/whitepapers/100005/cytokine-
quantification-in-drug-development.html). 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. PMID 23103935 (https://www.ncbi.
nlm.nih.gov/pubmed/23103935). doi:10.1038/nnano.2012.186 (https://doi.org/10.1038%2Fnnano.2012.186).
9. Schmidt, SD; Mazzella, MJ; Nixon, RA; Mathews, PM (2012). "A measurement by enzyme-linked
immunosorbent assay". Methods in Molecular Biology. 849: 50727. PMID 22528112 (https://www.ncbi.nl
m.nih.gov/pubmed/22528112). doi:10.1007/978-1-61779-551-0_34 (https://doi.org/10.1007%2F978-1-6177
9-551-0_34).
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:10.1186/2193-1801-2-
263 (https://doi.org/10.1186%2F2193-1801-2-263).
11. MedlinePlus Encyclopedia ELISA/Western blot tests for HIV (https://medlineplus.gov/ency/article/003538.ht
m)
12. "Food Allergen Partnership" (http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatory
Information/Allergens/ucm106779.htm) (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. PMID 10811336 (https://www.ncbi.nl
m.nih.gov/pubmed/10811336). doi:10.1111/j.1572-0241.2000.02018.x (https://doi.org/10.1111%2Fj.1572-02
41.2000.02018.x).
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. PMID 25385391 (http
s://www.ncbi.nlm.nih.gov/pubmed/25385391). doi:10.1002/jcla.21816 (https://doi.org/10.1002%2Fjcla.2181
6).
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)" (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1317074). Clinical and Vaccine
Immunology. 12 (12): 14019. PMC 1317074 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1317074) .
PMID 16339063 (https://www.ncbi.nlm.nih.gov/pubmed/16339063). doi:10.1128/CDLI.12.12.1401-
1409.2005 (https://doi.org/10.1128%2FCDLI.12.12.1401-1409.2005).

External links
More publications about ELISA kits (http://www.gentaurpub.com/elisa-kits/ZWxpc2Ega2l0cw==.html)
An animated illustration of an ELISA assay (http://www.immunospot.eu/elisa-animation.html)
The ELISA technique illustrated (https://www.mabtech.com/knowledge-center/assay-principles/elisa-assay-p
rinciple)
An animated tutorial comparing the direct and indirect ELISA methods (http://www.sumanasinc.com/webco
ntent/animations/content/ELISA.html)
"Introduction to ELISA Activity beginner walkthrough of ELISA used for detecting HIV, including
animations (http://www.biology.arizona.edu/immunology/activities/elisa/main.html) at University of Arizona
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A descriptive animation of the application of sandwich ELISA to Pregnancy test (http://www.whfreeman.co

m/catalog/static/whf/kuby/content/anm/kb07an01.htm)
Animated video overview of competitive ELISA formats (http://www.piercenet.com/method/overview-elisa#
elisaformats)
ELISA (https://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=&term=ELISA) at the US National Library
of Medicine Medical Subject Headings (MeSH)

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