Radioimmunoassay | |
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MeSH | D011863 |
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 concentrations (for example, hormone levels in blood) by use of antibodies.
The RIA technique is extremely sensitive and extremely specific, and although it requires specialized equipment, it remains among the least expensive methods to perform such measurements. It requires special precautions and licensing, since radioactive substances are used.[ citation needed ]
In contrast, an immunoradiometric assay (IRMA) is an immunoassay that uses radiolabeled molecules but in an immediate rather than stepwise way.
A radioallergosorbent test (RAST) is an example of radioimmunoassay. It is used to detect the causative allergen for an allergy.
Classically, to perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine, such as 125-I, or tritium [1] attached to tyrosine. This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two specifically bind to one another. Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added. This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody binding sites. As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated and the radioactivity of the free(unbound) antigen remaining in the supernatant is measured using a gamma counter. This value is then compared to a standardised calibration curve to work out the concentration of the unlabelled antigen in the patient serum sample. [2] RIAs can detect a few picograms of analyte in an experimental tubes if using antibodies of high affinity. [1]
This method can be used for any biological molecule in principle and is not restricted to serum antigens, nor is it required to use the indirect method of measuring the free antigen instead of directly measuring the captured antigen. For example, if it is undesirable or not possible to radiolabel the antigen or target molecule of interest, a RIA can be done if two different antibodies that recognize the target are available and the target is large enough (e.g., a protein) to present multiple epitopes to the antibodies. One antibody would be radiolabeled as above while the other would remain unmodified. The RIA would begin with the "cold" unlabeled antibody being allowed to interact and bind to the target molecule in solution. Preferably, this unlabeled antibody is immobilized in some way, such as coupled to an agarose bead, coated to a surface, etc. Next, the "hot" radiolabeled antibody is allowed to interact with the first antibody-target molecule complex. After extensive washing, the direct amount of radioactive antibody bound is measured and the amount of target molecule quantified by comparing it to a reference amount assayed at the same time. This method is similar in principle to the non-radioactive sandwich ELISA method. [3]
This method was developed by Solomon Berson and Rosalyn Sussman Yalow at the Veterans Administration Hospital in the Bronx, New York. [4] [5] This revolutionary development earned Dr. Yalow the Nobel Prize for Medicine in 1977, the second woman ever to win it. [6] In her acceptance speech, Dr. Yalow said, "The world cannot afford the loss of the talents of half its people if we are to solve the many problems which beset us." [7] Yalow shared the Nobel Prize with Roger Guillemin, and Andrew Schally who earned the prize based on their research into "the peptide hormone production of the brain". [6]
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 is a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand in a liquid sample using antibodies directed against the ligand to be measured. ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a quality control check in various industries.
Haptens are small molecules that elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself. The mechanisms of absence of immune response may vary and involve complex immunological interactions, but can include absent or insufficient co-stimulatory signals from antigen-presenting cells.
Rosalyn Sussman Yalow was an American medical physicist, and a co-winner of the 1977 Nobel Prize in Physiology or Medicine for development of the radioimmunoassay technique. She was the second woman, and the first American-born woman, to be awarded the Nobel Prize in Physiology or Medicine.
An immunoassay (IA) 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 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 are frequently measured using immunoassays for medical and research purposes.
An enzyme immunoassay is any of several immunoassay methods that use an enzyme bound to an antigen or antibody. These may include:
The hook effect refers to the prozone phenomenon, also known as antibody excess, or the postzone phenomenon, also known as antigen excess. It is an immunologic phenomenon whereby the effectiveness of antibodies to form immune complexes can be impaired when concentrations of an antibody or an antigen are very high. The formation of immune complexes stops increasing with greater concentrations and then decreases at extremely high concentrations, producing a hook shape on a graph of measurements. An important practical relevance of the phenomenon is as a type of interference that plagues certain immunoassays and nephelometric assays, resulting in false negatives or inaccurately low results. Other common forms of interference include antibody interference, cross-reactivity and signal interference. The phenomenon is caused by very high concentrations of a particular analyte or antibody and is most prevalent in one-step (sandwich) immunoassays.
Hemagglutination, or haemagglutination, is a specific form of agglutination that involves red blood cells (RBCs). It has two common uses in the laboratory: blood typing and the quantification of virus dilutions in a haemagglutination assay.
Solomon Aaron Berson was an American physician and scientist whose discoveries, mostly together with Rosalyn Yalow, caused major advances in clinical biochemistry. Five years after Berson's death, Yalow received a Nobel Prize, which cannot be awarded posthumously, for their joint work on the radioimmunoassay.
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 need for specialized and costly equipment. LFTs are widely used in medical diagnostics in the home, at the point of care, and in the laboratory. For instance, the home pregnancy test is an LFT that detects a specific hormone. These tests are simple and economical and generally show results in around five to thirty minutes. Many lab-based applications increase the sensitivity of simple LFTs by employing additional dedicated equipment. Because the target substance is often a biological antigen, many lateral flow tests are rapid antigen tests.
Anti-double stranded DNA (Anti-dsDNA) antibodies are a group of anti-nuclear antibodies (ANA) the target antigen of which is double stranded DNA. Blood tests such as enzyme-linked immunosorbent assay (ELISA) and immunofluorescence are routinely performed to detect anti-dsDNA antibodies in diagnostic laboratories. They are highly diagnostic of systemic lupus erythematosus (SLE) and are implicated in the pathogenesis of lupus nephritis.
Magnetic immunoassay (MIA) is a type of diagnostic immunoassay using magnetic beads as labels in lieu of conventional enzymes (ELISA), radioisotopes (RIA) or fluorescent moieties to detect a specified analyte. MIA involves the specific binding of an antibody to its antigen, where a magnetic label is conjugated to one element of the pair. The presence of magnetic beads is then detected by a magnetic reader (magnetometer) which measures the magnetic field change induced by the beads. The signal measured by the magnetometer is proportional to the analyte concentration in the initial sample.
A radiobinding assay is a method of detecting and quantifying antibodies targeted toward a specific antigen. As such, it can be seen as the inverse of radioimmunoassay, which quantifies an antigen by use of corresponding antibodies.
Roger Ekins FRS was a British biophysicist and professor at University College London. He was awarded the 1998 Edwin F. Ullman Award.
Immunoradiometric assay (IRMA) is an assay that uses radiolabeled antibodies. It differs from conventional radioimmunoassay (RIA) in that the compound to be measured combines immediately with the radiolabeled antibodies, rather than displacing another antigen by degrees over some period. Principle:- A noncompetitive assay in which analyte to be measured sandwich btw two Antibodies
Mass spectrometric immunoassay (MSIA) is a rapid method is used to detect and/ or quantify antigens and or antibody analytes. This method uses an analyte affinity isolation to extract targeted molecules and internal standards from biological fluid in preparation for matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). This method allows for "top down" and "bottom up" analysis. This sensitive method allows for a new and improved process for detecting multiple antigens and antibodies in a single assay. This assay is also capable of distinguishing mass shifted forms of the same molecule via a panantibody, as well as distinguish point mutations in proteins. Each specific form is detected uniquely based on their characteristic molecular mass. MSIA has dual specificity because of the antibody-antigen reaction coupled with the power of a mass spectrometer.
A ligand binding assay (LBA) is an assay, or an analytic procedure, which relies on the binding of ligand molecules to receptors, antibodies or other macromolecules. A detection method is used to determine the presence and amount of the ligand-receptor complexes formed, and this is usually determined electrochemically or through a fluorescence detection method. This type of analytic test can be used to test for the presence of target molecules in a sample that are known to bind to the receptor.
Shimon (Seymour) M. Glick is an American-born Israeli physician.
Ekkehard Kallee was a German university professor and doctor for nuclear medicine.
Fluorescence polarization immunoassay (FPIA) is a class of in vitro biochemical test used for rapid detection of antibody or antigen in sample. FPIA is a competitive homogenous assay, that consists of a simple prepare and read method, without the requirement of separation or washing steps.
The James J. Peters VA Medical Center,, is a US Department of Veterans Affairs hospital complex located at 130 West Kingsbridge Road in West Fordham, Bronx, New York City. The hospital is the headquarters of the Veterans Integrated Service Networks New York/New Jersey VA Health Care Network. This network is also the parent network to VA New York Harbor Healthcare System.
steps in radioimmunoassay technique