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Immunoassay
An immunoassay is a biochemical test that measures
Immunoassay
the presence or concentration of a macromolecule or a
small molecule in a solution through the use of an
antibody (usually) or an antigen (sometimes). The
molecule detected by the immunoassay is often referred to
as an "analyte" and is in many cases a protein, although it
may be other kinds of molecules, of different size and
types, as long as the proper antibodies that have the
adequate properties for the assay are developed. Analytes
in biological liquids such as serum or urine are frequently
measured using immunoassays for medical and research
purposes.[1]

Immunoassays come in many different formats and

variations. Immunoassays may be run in multiple steps
with reagents being added and washed away or separated
at different points in the assay. Multi-step assays are often
called separation immunoassays or heterogeneous
Illustration of the basic components of an
immunoassays. Some immunoassays can be carried out
immunoassay, which includes an analyte
simply by mixing the reagents and sample and making a
(green), an antibody (black), and a
physical measurement. Such assays are called
detectable label (yellow).
homogeneous immunoassays, or less frequently non-
separation immunoassays. MeSH D007118

The use of a calibrator is often employed in immunoassays. Calibrators are solutions that are known
to contain the analyte in question, and the concentration of that analyte is generally known.
Comparison of an assay's response to a real sample against the assay's response produced by the
calibrators makes it possible to interpret the signal strength in terms of the presence or concentration
of analyte in the sample.

Contents
Principle
History
Labels
Enzymes
Radioactive isotopes
DNA reporters
Fluorogenic reporters
Electrochemiluminescent tags

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Label-free immunoassays
Classifications and formats
Competitive, homogeneous immunoassays
Competitive, heterogeneous immunoassays
One-site, noncompetitive immunoassays
Two-site, noncompetitive immunoassays
Examples
Clinical tests
Sports anti-doping analysis
Research
Photoacoustic Immunoassay
See also
References
External links

Principle
Immunoassays rely on the ability of an antibody to recognize and bind a specific macromolecule in
what might be a complex mixture of macromolecules. In immunology the particular macromolecule
bound by an antibody is referred to as an antigen and the area on an antigen to which the antibody
binds is called an epitope.

In some cases, an immunoassay may use an antigen to detect for the presence of antibodies, which
recognize that antigen, in a solution. In other words, in some immunoassays, the analyte may be an
antibody rather than an antigen.

In addition to the binding of an antibody to its antigen, the other key feature of all immunoassays is a
means to produce a measurable signal in response to the binding. Most, though not all,
immunoassays involve chemically linking antibodies or antigens with some kind of detectable label. A
large number of labels exist in modern immunoassays, and they allow for detection through different
means. Many labels are detectable because they either emit radiation, produce a color change in a
solution, fluoresce under light, or can be induced to emit light.

History
Rosalyn Sussman Yalow and Solomon Berson are credited with the development of the first
immunoassays in the 1950s. Yalow accepted the Nobel Prize for her work in immunoassays in 1977,
becoming the second American woman to have won the award.[2]

Immunoassays became considerably simpler to perform and more popular when techniques for
chemically linked enzymes to antibodies were demonstrated in the late 1960s.[3]

In 1983, Professor Anthony Campbell[4] at Cardiff University replaced radioactive iodine used in
immunoassay with an acridinium ester that makes its own light: chemiluminescence. This type of
immunoassay is now used in around 100 million clinical tests every year worldwide, enabling
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clinicians to measure a wide range of proteins, pathogens and other molecules in blood samples.[5]

By 2012, the commercial immunoassay industry earned US$17,000,000,000 and was thought to have
prospects of slow annual growth in the 2 to 3 percent range.[6]

Labels
Immunoassays employ a variety of different labels to allow for detection of antibodies and antigens.
Labels are typically chemically linked or conjugated to the desired antibody or antigen.

Enzymes
Possibly one of the most popular labels to use in immunoassays is
enzymes. Immunoassays which employ enzymes are referred to
as enzyme-linked immunosorbent assays (ELISAs), or sometimes
enzyme immunoassays (EIAs).

Enzymes used in ELISAs include horseradish peroxidase (HRP),

alkaline phosphatase (AP) or glucose oxidase. These enzymes A sandwich ELISA run on a
microtitre plate.
allow for detection often because they produce an observable
color change in the presence of certain reagents. In some cases
these enzymes are exposed to reagents which cause them to produce light or Chemiluminescence.

Radioactive isotopes
Radioactive isotopes can be incorporated into immunoassay reagents to produce a radioimmunoassay
(RIA). Radioactivity emitted by bound antibody-antigen complexes can be easily detected using
conventional methods.

RIAs were some of the earliest immunoassays developed, but have fallen out of favor largely due to
the difficulty and potential dangers presented by working with radioactivity.[7][8]

DNA reporters
A newer approach to immunoassays involves combining real-time quantitative polymerase chain
reaction (RT qPCR) and traditional immunoassay techniques. Called real-time immunoquantitative
PCR (iqPCR) the label used in these assays is a DNA probe.[9][10]

Fluorogenic reporters
Fluorogenic reporters like phycoerythrin are used in a number of modern immunoassays.[11] Protein
microarrays are a type of immunoassay that often employ fluorogenic reporters.[12]

Electrochemiluminescent tags
Some labels work via electrochemiluminescence, in which the label emits detectable light in response
to electric current.[13]

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Label-free immunoassays
While some kind of label is generally employed in immunoassays, there are certain kinds of assays
which do not rely on labels, but instead employ detection methods that don't require the modification
or labeling the components of the assay. Surface plasmon resonance is an example of technique that
can detect binding between an unlabeled antibody and antigens.[14] Another demonstrated labeless
immunoassay involves measuring the change in resistance on an electrode as antigens bind to it.[15]

Classifications and formats

Immunoassays can be run in a number of different formats.
Generally, an immunoassay will fall into one of several categories
depending on how it is run.[16]

Competitive, homogeneous immunoassays

In a competitive, homogeneous immunoassay, unlabelled analyte
in a sample competes with labelled analyte to bind an antibody.
The amount of labelled, unbound analyte is then measured. In
theory, the more analyte in the sample, the more labelled analyte
gets displaced and then measured; hence, the amount of labelled,
unbound analyte is proportional to the amount of analyte in the
sample.

Competitive,
In a competitive, homogeneous
heterogeneous immunoassay unlabeled analyte
immunoassays displaces bound labelled analyte,
which is then detected or measured.
As in a competitive,
homogeneous immunoassay,
unlabelled analyte in a sample competes with labelled analyte to
bind an antibody. In the heterogeneous assays, the labelled,
unbound analyte is separated or washed away, and the remaining
Two-site, noncompetitive
immunoassays usually consist of an
labelled, bound analyte is measured.
analyte "sandwiched" between two
antibodies. ELISAs are often run in
this format
One-site, noncompetitive immunoassays
The unknown analyte in the sample binds with labelled
antibodies. The unbound, labelled antibodies are washed away,
and the bound, labelled antibodies are measured. The intensity of the signal is directly proportional to
the amount of unknown analyte.

Two-site, noncompetitive immunoassays

The analyte in the unknown sample is bound to the antibody site, then the labelled antibody is bound
to the analyte. The amount of labelled antibody on the site is then measured. It will be directly
proportional to the concentration of the analyte because the labelled antibody will not bind if the
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analyte is not present in the unknown sample. This type of immunoassay is also known as a sandwich
assay as the analyte is "sandwiched" between two antibodies.

Examples

Clinical tests
A wide range of medical tests are immunoassays, called immunodiagnostics in this context. Many
home pregnancy tests are immunoassays, which detect the pregnancy marker human chorionic
gonadotropin.[17][18] Other clinical immunoassays include tests that measure levels of CK-MB to
assess heart disease, insulin to assess hypoglycemia, prostate-specific antigen to detect prostate
cancer, and some are also used for the detection and/or quantitative measurement of some
pharmaceutical compounds (see Enzyme multiplied immunoassay technique for more details).[19]

Sports anti-doping analysis

Immunoassays are used in sports anti-doping laboratories to test athletes' blood samples for
prohibited recombinant human growth hormone (rhGH, rGH, hGH, GH). [20]

Research

Photoacoustic Immunoassay
The photoacoustic immunoassay measures low-frequency acoustic signals generated by metal
nanoparticle tags. Illuminated by a modulated light at a plasmon resonance wavelength, the
nanoparticles generate strong acoustic signal, which can be measured using a microphone.[21] The
photoacoustic immunoassay can be applied to lateral flow tests, which use colloidal nanoparticles.[22]

See also
ELISA
MELISA
CEDIA
Immunoscreening
Lateral flow test
Magnetic immunoassay
Radioimmunoassay
Surround Optical Fiber Immunoassay (SOFIA)
CD/DVD based immunoassay
Agglutination-PCR
Optical immunoassay

References
1. Yetisen A. K. (2013). "Paper-based microfluidic point-of-care diagnostic devices". Lab on a Chip.
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13 (12): 2210–2251. doi:10.1039/C3LC50169H (https://doi.org/10.1039%2FC3LC50169H).

PMID 23652632 (https://pubmed.ncbi.nlm.nih.gov/23652632).
2. Rall JE. Solomon A. Berson. In "Biographical Memoirs". National Academy of Sciences
1990;59:54-71. ISBN 0-309-04198-8. Fulltext (http://www.nap.edu/books/0309041988/html/54.htm
l).
3. Lequin R (2005). "Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA)".
Clin. Chem. 51 (12): 2415–8. doi:10.1373/clinchem.2005.051532 (https://doi.org/10.1373%2Fclinc
hem.2005.051532). PMID 16179424 (https://pubmed.ncbi.nlm.nih.gov/16179424).
4. Prof Anthony Campbell - MA PhD (http://www.cf.ac.uk/phrmy/contactsandpeople/fulltimeacademic
staff/campbell-anthonynew-biography_new.html), Cardiff University, retrieved 29 December 2012
5. "NPS Focus" (http://www.rsc.org/chemistryworld/Issues/2003/June/rainbow.asp), Rainbow
makers, Royal Society of Chemistry (RSC), 2003, retrieved 29 December 2012
6. Carlson, Bruce (15 February 2014). "Seizing Immunoassay Opportunities" (http://www.genengnew
s.com/gen-articles/seizing-immunoassay-opportunities/5135/). Gen. Eng. Biotechnol. News. 34
(4). pp. 12–13.
7. Susan J. Landers (3 April 2006). "ELISA test marks 35 years of answering medical questions" (htt
p://www.ama-assn.org/amednews/2006/04/03/hlsb0403.htm). American Medical News. Retrieved
9 December 2012.
8. "Rosalyn Sussman Yalow" (https://www.america.gov/st/diversity-english/2008/April/200804271350
47eaifas0.9798045.html#ixzz0rKOY0N87). America.gov. April 27, 2008. Retrieved June 26, 2010.
9. Rajkovic; El-Moualij (2006). "Immunoquantitative real-time PCR for detection and quantification of
Staphylococcus aureus enterotoxin B in foods" (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC16
10299). Applied and Environmental Microbiology. 72 (10): 6593–9. doi:10.1128/AEM.03068-05 (ht
tps://doi.org/10.1128%2FAEM.03068-05). PMC 1610299 (https://www.ncbi.nlm.nih.gov/pmc/article
s/PMC1610299). PMID 17021210 (https://pubmed.ncbi.nlm.nih.gov/17021210).
10. Gofflot; El (2004). "Immuno-quantitative polymerase chain reaction for detection and quantitation
of prion protein". Journal of Immunoassay and Immunochemistry. 25 (3): 241–58. doi:10.1081/ias-
200028044 (https://doi.org/10.1081%2Fias-200028044). PMID 15461386 (https://pubmed.ncbi.nl
m.nih.gov/15461386).
11. "Luminex xMAP Technology" (http://www.millipore.com/bmia/flx4/bmia_instruments&tab1=1&tab2
=2#tab2=2:tab1=1). Millipore Corporation. Retrieved 13 December 2012.
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(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2533396). PLOS ONE. 3 (9): e3265.
Bibcode:2008PLoSO...3.3265C (https://ui.adsabs.harvard.edu/abs/2008PLoSO...3.3265C).
doi:10.1371/journal.pone.0003265 (https://doi.org/10.1371%2Fjournal.pone.0003265).
PMC 2533396 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2533396). PMID 18813342 (https://
pubmed.ncbi.nlm.nih.gov/18813342).
13. Forster RJ, Bertoncello P, Keyes TE (2009). "Electrogenerated Chemiluminescence". Annual
Review of Analytical Chemistry. 2: 359–85. Bibcode:2009ARAC....2..359F (https://ui.adsabs.harva
rd.edu/abs/2009ARAC....2..359F). doi:10.1146/annurev-anchem-060908-155305 (https://doi.org/1
0.1146%2Fannurev-anchem-060908-155305). PMID 20636067 (https://pubmed.ncbi.nlm.nih.gov/
20636067).
14. J. B. González-Díaz; et al. (2008). "Plasmonic Au/Co/Au nanosandwiches with Enhanced
Magneto-Optical Activity". Small. 4 (2): 202–5. doi:10.1002/smll.200700594 (https://doi.org/10.100
2%2Fsmll.200700594). hdl:10261/17402 (https://hdl.handle.net/10261%2F17402).
PMID 18196506 (https://pubmed.ncbi.nlm.nih.gov/18196506).
15. Georgios Tsekenis (2008). "Label-free immunosensor assay for myelin basic protein based upon
an ac impedance protocol". Analytical Chemistry. 80 (6): 2058–62. doi:10.1021/ac702070e (http
s://doi.org/10.1021%2Fac702070e). PMID 18260654
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16. Goldys, Ewa (2009-08-24). Fluorescence Applications in Biotechnology and Life Sciences (https://
books.google.com/?id=4Lu_Fsspx9YC&printsec=frontcover&dq=Fluorescence+Applications+in+B
iotechnology+and+Life+Sciences). Wiley-Blackwell. p. 311. ISBN 978-0470083703. Retrieved
11 December 2012.
17. "ELISA for Home Pregnancy Test" (http://www.elisa-antibody.com/index.php?page=home-pregnan
cy-test). Retrieved 11 December 2012.
18. Butler, SA (2001). "Detection of early pregnancy forms of human chorionic gonadotropin by home
pregnancy test devices". Clinical Chemistry. 47 (12): 2131–6. PMID 11719477 (https://pubmed.nc
bi.nlm.nih.gov/11719477).
19. Twyman, SA (2001). "Immunoassays, applications: Clinica" (https://web.archive.org/web/2012032
4055803/http://www.writescience.com/RMT%20PDFs/Elsevier/eans%20immoassays%20clinapp.
pdf) (PDF). Encyclopedia of Analytical Science. 4: 317–324. Archived from the original (http://ww
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March 2012. Retrieved 11 December 2012.
20. Tsivou M; Kioukia-Fougia N; Lyris E; Aggelis Y; Fragkaki A; Kiousi X; Simitsek Ph; Dimopoulou H;
Leontiou I-P; Stamou M; Spyridaki M-H; Georgakopoulos C (2006). "An overview of the doping
control analysis during the Olympic Games of 2004 in Athens, Greece". Analytica Chimica Acta.
555: 1–13. doi:10.1016/j.aca.2005.08.068 (https://doi.org/10.1016%2Fj.aca.2005.08.068).
21. Zhao Y, Cao M, McClelland JF, Lu M (2016). "A photoacoustic immunoassay for biomarker
detection". Biosensors and Bioelectronics. 85: 261–66. doi:10.1016/j.bios.2016.05.028 (https://doi.
org/10.1016%2Fj.bios.2016.05.028). PMID 27183276 (https://pubmed.ncbi.nlm.nih.gov/2718327
6).
22. Zhao Y, Huang Y, Zhao X, McClelland JF, Lu M (2016). "Nanoparticle-based photoacoustic
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External links
"The Immunoassay Handbook", 3rd Edition, David Wild, Ed., Elsevier,2008

Immunoassay (https://meshb.nlm.nih.gov/record/ui?name=Immunoassay) at the US National

Library of Medicine Medical Subject Headings (MeSH)
Chapter 5 and 6 in the book "Bioanalytical Chemistry" by Susan R. Mikkelsen

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