Immunoassay

Immunoassay
Error creating thumbnail: sh: /usr/local/bin/rsvg-convert: not found Illustration of the basic components of an immunoassay, which includes an analyte (green), an antibody (black), and a detectable label (yellow).
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MeSH	D007118

An immunoassay is a biochemical test that measures the presence or concentration of a macromolecule in a solution through the use of an antibody or immunoglobulin. The macromolecule detected by the immunoassay is often referred to as an "analyte" and is in many cases a protein. Analytes in biological liquids such as serum or urine 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 homogenous 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 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 because they 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 would accept the Nobel Prize for her work in immunoassays in 1977, becoming the second American woman to have won the award.

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 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 US$17 billion 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.

A sandwich ELISA run on a microtitre plate.

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 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.

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 reporters like phycoerythrin are used in a number of modern immunoassays. Protein microarrays are a type of immunoassay that often employ flourogenic reporters.

Electrochemiluminescent tags

Some labels work on an electrochemiluminescent principle, in which the label emits detectable light in response to electric current.

Labeless 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. Another demonstrated labeless immunoassay involves measuring the change in resistance on an electrode as antigen binds to it.

Classifications and formats

In a competitive, homogeneous immunoassay unlabeled analyte displaces bound labelled analyte, which is then detected or measured.

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 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 competed off and hence the amount of labelled, unbound analyte is proportional to the amount of analyte in the sample.

Two-site, noncompetitive immunoassays usually consist of an analyte "sandwiched" between two antibodies. ELISAs are often run in this format

Competitive, heterogeneous immunoassays

As in a competitive, heterogeneous 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 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 labelled antibody will not bind if the analyte is not present in the unknown sample. This type is also known as sandwich assay as the analyte is "sandwiched" between two antibodies.

Examples

Clinical tests

A wide range of clinical tests are immunoassays. Many home pregnancy tests are immunoassays, which detect the pregnancy marker human chorionic gonadotropin. A few examples of other clinical immunoassays include tests that measure levels of CK-MB to assess heart disease, insulin to assess hypoglycemia and prostate-specific antigen to detect prostate cancer.

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). B Bonds

See also

• ELISA
• MELISA
• CEDIA
• Immunoscreening
• Lateral flow test
• Magnetic immunoassay
• Radioimmunoassay
• Surround Optical Fiber Immunoassay (SOFIA)
• CD/DVD based immunoassay

External links

"The Immunoassay Handbook", 3rd Edition, David Wild, Ed., Elsevier,2008

• Learning Guides from Abbott Laboratories on Immunoassay (English): Part 1 Part 2 Part 3 Part 4 Appendix
• Immunoassay at the US National Library of Medicine Medical Subject Headings (MeSH)
• Automation of Immunoassays
• Chapter 5 and 6 in the book "Bioanalytical Chemistry" by Susan R. Mikkelsen