An immunoassay is a biochemical test that measures 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.
Illustration of the basic components of an immunoassay, which includes an analyte (green), an antibody (black), and a detectable label (yellow).
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.
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.
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.
In 1983, Professor Anthony Campbell 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 clinicians to measure a wide range of proteins, pathogens and other molecules in blood samples.
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.
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 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 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.
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.
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. Another demonstrated labeless immunoassay involves measuring the change in resistance on an electrode as antigens bind to it.
Classifications and formats
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, 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.
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. 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).
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.
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