Radioimmunoassay
Encyclopedia
Radioimmunoassay is a very sensitive in vitro
assay
technique used to measure concentrations of antigen
s (for example, hormone
levels in the blood
) by use of antibodies. As such, it can be seen as the inverse of a radiobinding assay
, which quantifies an antibody by use of corresponding antigens.
Although the RIA technique is extremely sensitive and extremely specific, requiring specialized equipment, it remains the least expensive method to perform such tests. It requires special precautions and licensing, since radioactive substances are used. Today it has been supplanted by the ELISA
method, where the antigen-antibody reaction is measured using colorimetric
signals instead of a radioactive signal. However, because of its robustness, consistent results and low price per test , RIA methods are again becoming popular. It is generally more simple to perform than a bioassay
.
The RAST test
(radioallergosorbent test) is an example of radioimmunoassay. It is used to detect the causative allergen
for an allergy
.
is made radioactive, frequently by labeling it with gamma-radioactive isotope
s of iodine
attached to tyrosine
. This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two chemically 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 from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter
. Using known standards, a binding curve can then be generated which allows the amount of antigen in the patient's serum to be derived.
working at the Bronx Veterans Administration Hospital affiliated with the Mount Sinai School of Medicine
. In 1977, Rosalyn Sussman Yalow
received the Nobel Prize in Medicine for the development of the RIA for insulin: the precise measurement of minute amounts of such a hormone
revolutionized the field of endocrinology
. By allowing the precise measurement of blood levels of hormones the mechanism of hormone deficiency diseases could be identified, and better treated. Yalow and Berson refused to patent the assay, because they felt that it should be freely available to the field of medicine. Berson would have shared the prize, but he had died before the award was given.
With this technique, separating bound from unbound antigen is crucial. Initially, the method of separation employed was the use of a second "anti-antibody" directed against the first for precipitation and centrifugation. The use of charcoal suspension for precipitation was extended but replaced later by Drs. Werner and Acebedo at Columbia University for RIA of T3 and T4. An ultramicro RIA for human TSH was published in BBRC (1975) by Drs. Acebedo, Hayek et al.
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
assay
Assay
An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...
technique used to measure concentrations of antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
s (for example, hormone
Hormone
A hormone is a chemical released by a cell or a gland in one part of the body that sends out messages that affect cells in other parts of the organism. Only a small amount of hormone is required to alter cell metabolism. In essence, it is a chemical messenger that transports a signal from one...
levels in the blood
Blood
Blood is a specialized bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells....
) by use of antibodies. As such, it can be seen as the inverse of a radiobinding assay
Radiobinding assay
A radiobinding assay is a method of detecting and quantifying antibodies targeted towards a specific antigen. As such, it can be seen as the inverse of radioimmunoassay, which quantifies an antigen by use of corresponding antibodies.-Technique:...
, which quantifies an antibody by use of corresponding antigens.
Although the RIA technique is extremely sensitive and extremely specific, requiring specialized equipment, it remains the least expensive method to perform such tests. It requires special precautions and licensing, since radioactive substances are used. Today it has been supplanted by the ELISA
ELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
method, where the antigen-antibody reaction is measured using colorimetric
Colorimetry
Colorimetry is "the science and technology used to quantify and describe physically the human color perception."It is similar to spectrophotometry, but is distinguished by its interest in reducing spectra to the physical correlates of color perception, most often the CIE 1931 XYZ color space...
signals instead of a radioactive signal. However, because of its robustness, consistent results and low price per test , RIA methods are again becoming popular. It is generally more simple to perform than a bioassay
Bioassay
Bioassay , or biological standardization is a type of scientific experiment. Bioassays are typically conducted to measure the effects of a substance on a living organism and are essential in the development of new drugs and in monitoring environmental pollutants...
.
The RAST test
RAST test
A RAST test is a blood test used to determine to what substances a person is allergic. This is different from a skin allergy test, which determines allergy by the reaction of a person's skin to different substances....
(radioallergosorbent test) is an example of radioimmunoassay. It is used to detect the causative allergen
Allergen
An allergen is any substance that can cause an allergy. In technical terms, an allergen is a non-parasitic antigen capable of stimulating a type-I hypersensitivity reaction in atopic individuals....
for an allergy
Allergy
An Allergy is a hypersensitivity disorder of the immune system. Allergic reactions occur when a person's immune system reacts to normally harmless substances in the environment. A substance that causes a reaction is called an allergen. These reactions are acquired, predictable, and rapid...
.
Method
To perform a radioimmunoassay, a known quantity of an antigenAntigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
is made radioactive, frequently by labeling it with gamma-radioactive isotope
Isotope
Isotopes are variants of atoms of a particular chemical element, which have differing numbers of neutrons. Atoms of a particular element by definition must contain the same number of protons but may have a distinct number of neutrons which differs from atom to atom, without changing the designation...
s of iodine
Iodine
Iodine is a chemical element with the symbol I and atomic number 53. The name is pronounced , , or . The name is from the , meaning violet or purple, due to the color of elemental iodine vapor....
attached to tyrosine
Tyrosine
Tyrosine or 4-hydroxyphenylalanine, is one of the 22 amino acids that are used by cells to synthesize proteins. Its codons are UAC and UAU. It is a non-essential amino acid with a polar side group...
. This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two chemically bind to one another. Then, a sample of serum
Blood serum
In blood, the serum is the component that is neither a blood cell nor a clotting factor; it is the blood plasma with the fibrinogens removed...
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
Concentration
In chemistry, concentration is defined as the abundance of a constituent divided by the total volume of a mixture. Four types can be distinguished: mass concentration, molar concentration, number concentration, and volume 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 from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter
Gamma counter
A Gamma Counter is a machine to measure gamma radiation emitted by a radionuclide. Up to 300 samples are placed in sealed vials or test tubes, and move along a serpentine track on a horizontal plane. One at a time, they move down inside a shielded detector, set to measure specific energy windows...
. Using known standards, a binding curve can then be generated which allows the amount of antigen in the patient's serum to be derived.
History
It was developed in the 1950s by Rosalyn Yalow and Solomon Aaron BersonSolomon Berson
Solomon Aaron Berson was an American physician and scientist whose discoveries, mostly together with Rosalyn Yalow, caused major advances in clinical biochemistry....
working at the Bronx Veterans Administration Hospital affiliated with the Mount Sinai School of Medicine
Mount Sinai School of Medicine
Mount Sinai School of Medicine is an American medical school in the borough of Manhattan in New York City, currently ranked among the top 20 medical schools in the United States. It was chartered by Mount Sinai Hospital in 1963....
. In 1977, Rosalyn Sussman Yalow
Rosalyn Sussman Yalow
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...
received the Nobel Prize in Medicine for the development of the RIA for insulin: the precise measurement of minute amounts of such a hormone
Hormone
A hormone is a chemical released by a cell or a gland in one part of the body that sends out messages that affect cells in other parts of the organism. Only a small amount of hormone is required to alter cell metabolism. In essence, it is a chemical messenger that transports a signal from one...
revolutionized the field of endocrinology
Endocrinology
Endocrinology is a branch of biology and medicine dealing with the endocrine system, its diseases, and its specific secretions called hormones, the integration of developmental events such as proliferation, growth, and differentiation and the coordination of...
. By allowing the precise measurement of blood levels of hormones the mechanism of hormone deficiency diseases could be identified, and better treated. Yalow and Berson refused to patent the assay, because they felt that it should be freely available to the field of medicine. Berson would have shared the prize, but he had died before the award was given.
With this technique, separating bound from unbound antigen is crucial. Initially, the method of separation employed was the use of a second "anti-antibody" directed against the first for precipitation and centrifugation. The use of charcoal suspension for precipitation was extended but replaced later by Drs. Werner and Acebedo at Columbia University for RIA of T3 and T4. An ultramicro RIA for human TSH was published in BBRC (1975) by Drs. Acebedo, Hayek et al.
External links
- Radioimmunoassay- procedure
- http://www.lib.mcg.edu/edu/esimmuno/ch4/radassay.htm
- http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/Radioimmunoassay.html