Immunoassay
Encyclopedia
An immunoassay is a biochemical test that measures the presence or concentration
of a substance in solutions that frequently contain a complex mixture of substances. Analyte
s in biological liquids such as serum
or urine
are frequently assayed using immunoassay methods. Such assays are based on the unique ability of an antibody
to bind with high specificity to one or a very limited group of molecules. A molecule that binds to an antibody
is called an antigen
. Immunoassays can be carried out for either member of an antigen/antibody pair. For antigen analytes, an antibody that specifically binds to that antigen can frequently be prepared for use as an analytical reagent. When the analyte is a specific antibody its cognate antigen can be used as the analytical reagent. In either case the specificity of the assay depends on the degree to which the analytical reagent is able to bind to its specific binding partner to the exclusion of all other substances that might be present in the sample to be analyzed. In addition to the need for specificity, a binding partner must be selected that has a sufficiently high affinity for the analyte to permit an accurate measurement. The affinity requirements depend on the particular assay format that is used.
In addition to binding specificity, the other key feature of all immunoassays is a means to produce a measurable signal in response to a specific binding. Historically this was accomplished by measuring a change in some physical characteristic such as light scattering or changes in refractive index. With modern instrumentation such methods are again becoming increasingly popular. Nevertheless most immunoassays today depend on the use of an analytical reagent that is associated with a detectable label. A large variety of labels have been demonstrated including radioactive elements used in radioimmunoassays; enzymes; fluorescent, phosphorescent, and chemiluminescent dyes; latex and magnetic particles; dye crystalites, gold, silver, and selenium colloidal particles; metal chelates; coenzymes; electroactive groups; oligonucleotides, stable radicals,and others. Such labels serve for detection and quantitation of binding events either after separating free and bound labeled reagents or by designing the system in such a way that a binding event effects a change in the signal produced by the label. Immunoassays requiring a separation step, often called separation immunoassays or heterogeneous immunoassays, are popular because they are easy to design, but they frequently require multiple steps including careful washing of a surface onto which the labeled reagent has bound. Immunoassays in which the signal is affected by binding can often be run without a separation step. Such assays can frequently 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.
Regardless of the method used, interpretation of the signal produced in an immunoassay requires reference to a calibrator that mimics the characteristics of the sample medium. For qualitative assays the calibrators may consist of a negative sample with no analyte and a positive sample having the lowest concentration of the analyte that is considered detectable. Quantitative assays require additional calibrators with known analyte concentrations. Comparison of the assay response of a real sample to the assay responses produced by the calibrators makes it possible to interpret the signal strength in terms of the presence or concentration of analyte in the sample.
in the unknown sample competes with labeled antigen
to bind with antibodies. The amount of unbound, labeled antigen
is then measured, which is directly proportional to the concentration of sample antigen
. This is because the presence of more free, labeled antigens means there was little to no binding sites available on the antibodies as they were all saturated with the sample antigen
, which means it's high in number.
in the unknown sample competes with labeled antigen
to bind with antibodies. The amount of labeled antigen
bound to the antibody
site is then measured. In this method, the response will be inversely related to the concentration of antigen
in the unknown. This is because the greater the response, the less antigen
in the unknown was available to compete with the labeled antigen
.
in the sample binds with labeled antibodies. The unbound, labeled antibodies are washed away, and the bound, labeled is measured, which is directly proportional to the amount of unknown antigen
.
in the unknown is bound to the antibody
site, then labeled antibody
is bound to the antigen
. The amount of labeled antibody
on the site is then measured. It will be directly proportional to the concentration of the antigen
because labeled antibody
will not bind if the antigen
is not present in the unknown sample. This type is also known as sandwich assay as the antigen
is "sandwiched" between two antibodies.
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 a substance in solutions that frequently contain a complex mixture of substances. Analyte
Analyte
An analyte, or component , is a substance or chemical constituent that is of interest in an analytical procedure. Grammatically, it is important to note that experiments always seek to measure properties of analytes—and that analytes themselves can never be measured. For instance, one cannot...
s in biological liquids such as serum
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...
or urine
Urine
Urine is a typically sterile liquid by-product of the body that is secreted by the kidneys through a process called urination and excreted through the urethra. Cellular metabolism generates numerous by-products, many rich in nitrogen, that require elimination from the bloodstream...
are frequently assayed using immunoassay methods. Such assays are based on the unique ability of an antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
to bind with high specificity to one or a very limited group of molecules. A molecule that binds to an antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
is called an 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...
. Immunoassays can be carried out for either member of an antigen/antibody pair. For antigen analytes, an antibody that specifically binds to that antigen can frequently be prepared for use as an analytical reagent. When the analyte is a specific antibody its cognate antigen can be used as the analytical reagent. In either case the specificity of the assay depends on the degree to which the analytical reagent is able to bind to its specific binding partner to the exclusion of all other substances that might be present in the sample to be analyzed. In addition to the need for specificity, a binding partner must be selected that has a sufficiently high affinity for the analyte to permit an accurate measurement. The affinity requirements depend on the particular assay format that is used.
In addition to binding specificity, the other key feature of all immunoassays is a means to produce a measurable signal in response to a specific binding. Historically this was accomplished by measuring a change in some physical characteristic such as light scattering or changes in refractive index. With modern instrumentation such methods are again becoming increasingly popular. Nevertheless most immunoassays today depend on the use of an analytical reagent that is associated with a detectable label. A large variety of labels have been demonstrated including radioactive elements used in radioimmunoassays; enzymes; fluorescent, phosphorescent, and chemiluminescent dyes; latex and magnetic particles; dye crystalites, gold, silver, and selenium colloidal particles; metal chelates; coenzymes; electroactive groups; oligonucleotides, stable radicals,and others. Such labels serve for detection and quantitation of binding events either after separating free and bound labeled reagents or by designing the system in such a way that a binding event effects a change in the signal produced by the label. Immunoassays requiring a separation step, often called separation immunoassays or heterogeneous immunoassays, are popular because they are easy to design, but they frequently require multiple steps including careful washing of a surface onto which the labeled reagent has bound. Immunoassays in which the signal is affected by binding can often be run without a separation step. Such assays can frequently 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.
Regardless of the method used, interpretation of the signal produced in an immunoassay requires reference to a calibrator that mimics the characteristics of the sample medium. For qualitative assays the calibrators may consist of a negative sample with no analyte and a positive sample having the lowest concentration of the analyte that is considered detectable. Quantitative assays require additional calibrators with known analyte concentrations. Comparison of the assay response of a real sample to the assay responses produced by the calibrators makes it possible to interpret the signal strength in terms of the presence or concentration of analyte in the sample.
Types
Classification of immunoassays depends on whether one (noncompetitive) or two (competitive) antigens are used in the test system:Competitive, homogenous immunoassay
The 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...
in the unknown sample competes with labeled 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...
to bind with antibodies. The amount of unbound, labeled 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...
is then measured, which is directly proportional to the concentration of sample 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...
. This is because the presence of more free, labeled antigens means there was little to no binding sites available on the antibodies as they were all saturated with the sample 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...
, which means it's high in number.
Competitive, heterogenous immunoassay
The 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...
in the unknown sample competes with labeled 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...
to bind with antibodies. The amount of labeled 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...
bound to the antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
site is then measured. In this method, the response will be inversely related to the concentration 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...
in the unknown. This is because the greater the response, the less 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...
in the unknown was available to compete with the labeled 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...
.
One-slit, noncompetitive immunoassay
The unknown 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...
in the sample binds with labeled antibodies. The unbound, labeled antibodies are washed away, and the bound, labeled is measured, which is directly proportional to the amount of unknown 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...
.
Two-slit, noncompetitive immunoassays
The 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...
in the unknown is bound to the antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
site, then labeled antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
is bound to the 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...
. The amount of labeled antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
on the site is then measured. It will be directly proportional to the concentration of the 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...
because labeled antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
will not bind if the 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...
is not present in the unknown sample. This type is also known as sandwich assay as the 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...
is "sandwiched" between two antibodies.
Clinical tests
Owing to their high sensitivity (ability to detect very small concentrations) and specificity (minimal to no cross-reactivity), immunoassays have been employed in the measurement of blood leves of vitamins, hormones, and porphyrins.Sports anti-doping analysis
Immunoassay is used in sports anti-doping laboratories to test athletes' blood samples for prohibited recombinant human growth hormone (rhGH, rGH, hGH, GH).See also
- ELISAELISAEnzyme-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...
- MELISAMELISAA MELISA test is a blood test that detects Type-IV allergy to metals, chemicals, environmental toxins and molds from one single blood sample. It can also identify active Lyme disease . The test does not measure toxicity-that is to say, it will not measure the amounts of a harmful substance in the...
- ImmunoscreeningImmunoscreeningImmunoscreening is a method of biotechnology to detect a polypeptide produced from a cloned gene....
- Lateral flow testLateral flow testLateral flow tests also known as Lateral Flow Immunochromatographic Assays are a simple device intended to detect the presence of a target analyte in sample . Most commonly these tests are used for medical diagnostics either for home testing, point of care testing, or laboratory use...
- Magnetic immunoassayMagnetic immunoassayMagnetic immunoassay is a novel type of diagnostic immunoassay using magnetic beads as labels in lieu of conventional enzymes , radioisotopes or fluorescent moieties . This assay involves the specific binding of an antibody to its antigen, where a magnetic label is conjugated to one element of...
- RadioimmunoassayRadioimmunoassayRadioimmunoassay is a very sensitive in vitro assay technique used to measure concentrations of antigens by use of antibodies...
- Surround Optical Fiber Immunoassay (SOFIA)Surround Optical Fiber Immunoassay (SOFIA)Surround Optical Fiber Immunoassay is an ultra-sensitive, in vitro diagnostic platform incorporating a surround optical fiber assembly that captures fluorescence emissions from an entire sample. The technology's defining characteristics are its extremely high limit of detection, sensitivity and...