An immunoassay (IA) is a
biochemical test that measures the presence or
concentration
In chemistry, concentration is the abundance of a constituent divided by the total volume of a mixture. Several types of mathematical description can be distinguished: '' mass concentration'', ''molar concentration'', '' number concentration'', ...
of a
macromolecule or a small molecule in a solution through the use of an
antibody
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
(usually) or an
antigen
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune respons ...
(sometimes). The molecule detected by the immunoassay is often referred to as an "
analyte
An analyte, component (in clinical chemistry), or chemical species is a substance or chemical constituent that is of interest in an analytical procedure. The purest substances are referred to as analytes, such as 24 karat gold, NaCl, water, etc. ...
" and is in many cases a
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
, although it may be other kinds of molecules, of different sizes and types, as long as the proper antibodies that have the required properties for the assay are developed. Analytes in biological liquids such as
serum or
urine
Urine is a liquid by-product of metabolism in humans and in many other animals. Urine flows from the kidneys through the ureters to the urinary bladder. Urination results in urine being excreted from the body through the urethra.
Cellular ...
are frequently measured using immunoassays for medical and research purposes.
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 immunoassays. Some immunoassays can be carried out simply by mixing the reagents and sample and making a physical measurement. Such assays are called homogeneous immunoassays, or less frequently non-separation immunoassays.
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.
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
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune respons ...
and the area on an antigen to which the antibody binds is called an
epitope
An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The epitope is the specific piece of the antigen to which an antibody binds. The p ...
.
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
Rosalyn Sussman Yalow (July 19, 1921 – May 30, 2011) was an American medical physicist, and a co-winner of the 1977 Nobel Prize in Physiology or Medicine (together with Roger Guillemin and Andrew Schally) for development of the radioimmunoassay ...
and
Solomon Berson
Solomon Aaron Berson (April 22, 1918 – April 11, 1972) was an American physician and scientist whose discoveries, mostly together with Rosalyn Yalow, caused major advances in clinical biochemistry.Rall JE. ''Solomon A. Berson''. In "Biographica ...
are credited with the development of the first immunoassays in the 1950s. Yalow accepted the
Nobel Prize
The Nobel Prizes ( ; sv, Nobelpriset ; no, Nobelprisen ) are five separate prizes that, according to Alfred Nobel's will of 1895, are awarded to "those who, during the preceding year, have conferred the greatest benefit to humankind." Alfr ...
for her work in immunoassays in 1977, becoming the second American woman to have won the award.
[Rall JE. ''Solomon A. Berson''. In "Biographical Memoirs". National Academy of Sciences 1990;59:54-71. ]
Fulltext
Immunoassays became considerably simpler to perform and more popular when techniques for chemically linked enzymes to antibodies were demonstrated in the late 1960s.
In 1983, Professor
Anthony Campbell
at
Cardiff University replaced
radioactive iodine
There are 37 known isotopes of iodine (53I) from 108I to 144I; all undergo radioactive decay except 127I, which is stable. Iodine is thus a monoisotopic element.
Its longest-lived radioactive isotope, 129I, has a half-life of 15.7 million year ...
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 clinicians to measure a wide range of proteins, pathogens and other molecules in blood samples.
By 2012, the commercial immunoassay industry earned and was thought to have prospects of slow annual growth in the 2 to 3 percent range.
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
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
s. Immunoassays which employ enzymes are referred to as enzyme immunoassays (EIAs), of which
enzyme-linked immunosorbent assay
The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence ...
s (ELISAs) and
enzyme multiplied immunoassay technique (EMIT) are the most common types.
Enzymes used in ELISAs include
horseradish peroxidase (HRP),
alkaline phosphatase (AP) or
glucose oxidase
The glucose oxidase enzyme (GOx or GOD) also known as notatin (EC number 1.1.3.4) is an oxidoreductase that catalyses the oxidation of glucose to hydrogen peroxide and D-glucono-δ-lactone. This enzyme is produced by certain species of fungi and ...
. These enzymes 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
isotope
Isotopes are two or more types of atoms that have the same atomic number (number of protons in their nuclei) and position in the periodic table (and hence belong to the same chemical element), and that differ in nucleon numbers (mass numb ...
s can be incorporated into immunoassay reagents to produce a
radioimmunoassay
A radioimmunoassay (RIA) is an immunoassay that uses radiolabeled molecules in a stepwise formation of immune complexes. A RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, usually measuring antigen conc ...
(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.
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.
Fluorogenic reporters
Fluorogenic
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
reporters like
phycoerythrin are used in a number of modern immunoassays.
Protein microarray
A protein microarray (or protein chip) is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale. Its main advantage lies in the fact that larg ...
s are a type of immunoassay that often employ fluorogenic reporters.
Electrochemiluminescent tags
Some labels work via
electrochemiluminescence
Electrochemiluminescence or electrogenerated chemiluminescence (ECL) is a kind of luminescence produced during electrochemical reactions in solutions. In electrogenerated chemiluminescence, electrochemically generated intermediates undergo a highl ...
(ECL), in which the label emits detectable light in response to electric current.
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 do not require the modification or labeling the components of the assay.
Surface plasmon resonance
Surface plasmon resonance (SPR) is the resonant oscillation of conduction electrons at the interface between negative and positive permittivity material in a particle stimulated by incident light. SPR is the basis of many standard tools for measu ...
is an example of technique that can detect binding between an unlabeled antibody and antigens. Another demonstrated labeless immunoassay involves measuring the change in resistance on an electrode as
antigens bind to it.
Classifications and formats
![Competitive homogeneous immunoassay](https://upload.wikimedia.org/wikipedia/commons/5/5c/Competitive_homogeneous_immunoassay.svg)
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.
Competitive, homogeneous immunoassays
In a competitive, homogeneous immunoassay, unlabelled analyte in a sample competes with labeled 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, heterogeneous immunoassays
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 labelled, bound analyte is measured.
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 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 test
A pregnancy test is used to determine whether a female is pregnant or not. The two primary methods are testing for the female pregnancy hormone (human chorionic gonadotropin (hCG)) in blood or urine using a pregnancy test kit, and scanning with ...
s are immunoassays, which detect the pregnancy marker
human chorionic gonadotropin
Human chorionic gonadotropin (hCG) is a hormone for the maternal recognition of pregnancy produced by trophoblast cells that are surrounding a growing embryo (syncytiotrophoblast initially), which eventually forms the placenta after implantatio ...
. Other clinical immunoassays include tests that measure levels of
CK-MB
The CPK-MB test (creatine phosphokinase-MB), also known as CK-MB test, is a cardiac marker used to assist diagnoses of an acute myocardial infarction, myocardial ischemia, or myocarditis. It measures the blood level of CK-MB (creatine kinase myoca ...
to assess heart disease,
insulin to assess
hypoglycemia
Hypoglycemia, also called low blood sugar, is a fall in blood sugar to levels below normal, typically below 70 mg/dL (3.9 mmol/L). Whipple's triad is used to properly identify hypoglycemic episodes. It is defined as blood glucose bel ...
,
prostate-specific antigen
Prostate-specific antigen (PSA), also known as gamma-seminoprotein or kallikrein-3 (KLK3), P-30 antigen, is a glycoprotein enzyme encoded in humans by the ''KLK3'' gene. PSA is a member of the kallikrein-related peptidase family and is secreted b ...
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).
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).
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. The photoacoustic immunoassay can be applied to lateral flow tests, which use colloidal nanoparticles.
See also
*
ELISA
The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presen ...
*
MELISA
MELISA (Memory Lymphocyte Immunostimulation Assay) is a blood test that detects type IV hypersensitivity to metals, chemicals, environmental toxins and molds. Type IV hypersensitivity reactions, particularly to nickel, are well established and ma ...
*
ECLIA
*
CEDIA
*
Immunoscreening
*
Nephelometry
A nephelometer or aerosol photometer is an instrument for measuring the concentration of suspended particulates in a liquid or gas colloid. A nephelometer measures suspended particulates by employing a light beam (source beam) and a light dete ...
*
Lateral flow test
A lateral flow test (LFT), is an assay also known as a lateral flow device (LFD), lateral flow immunochromatographic assay, or rapid test. It is a simple device intended to detect the presence of a target substance in a liquid sample without the ...
*
Magnetic immunoassay
*
Radioimmunoassay
A radioimmunoassay (RIA) is an immunoassay that uses radiolabeled molecules in a stepwise formation of immune complexes. A RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, usually measuring antigen conc ...
*
Surround Optical Fiber Immunoassay (SOFIA)
Surround optical-fiber immunoassay (SOFIA) is an ultrasensitive, ''in vitro'' diagnostic platform incorporating a surround optical-fiber assembly that captures fluorescence emissions from an entire sample. The technology's defining characteristi ...
*
CD/DVD based immunoassay
*
Agglutination-PCR
References
External links
"The Immunoassay Handbook", 3rd Edition, David Wild, Ed., Elsevier,2008
*
* Chapter 5 and 6 in the book "Bioanalytical Chemistry" by Susan R. Mikkelsen
{{Immunologic techniques and tests
Biochemistry methods
Immunologic tests