An
immunoassay is a biochemical test that measures the
concentrationIn chemistry, concentration is the measure of how much of a given substance there is mixed with another substance. This can apply to any sort of chemical mixture, but most frequently the concept is limited to homogeneous solutions, where it refers to the amount of solute in the solvent.To...
of a substance in a biological liquid, typically
serumBlood plasma is the yellow liquid component of blood, in which the blood cells in whole blood would normally be suspended. It makes up about 55% of the total blood volume. It is mostly water and contains dissolved proteins, glucose, clotting factors, mineral ions, hormones and carbon dioxide...
or
urineUrine is a liquid waste product of the body secreted by the kidneys by a process of filtration from blood called urination and excreted through the urethra. Cellular metabolism generates numerous waste compounds, many rich in nitrogen, that require elimination from the bloodstream...
, using the reaction of an
antibodyAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
or antibodies to its
antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
. The
assayAn assay is a procedure in molecular biology for testing and/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...
takes advantage of the specific binding of an
antibodyAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
to its
antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
.
Monoclonal antibodiesMonoclonal antibodies are monospecific antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent cell. Given almost any substance, it is possible to create monoclonal antibodies that specifically bind to that substance; they can then...
are often used as they only usually bind to one site of a particular molecule, and therefore provide a more specific and accurate test, which is less easily confused by the presence of other molecules.
An
immunoassay is a biochemical test that measures the
concentrationIn chemistry, concentration is the measure of how much of a given substance there is mixed with another substance. This can apply to any sort of chemical mixture, but most frequently the concept is limited to homogeneous solutions, where it refers to the amount of solute in the solvent.To...
of a substance in a biological liquid, typically
serumBlood plasma is the yellow liquid component of blood, in which the blood cells in whole blood would normally be suspended. It makes up about 55% of the total blood volume. It is mostly water and contains dissolved proteins, glucose, clotting factors, mineral ions, hormones and carbon dioxide...
or
urineUrine is a liquid waste product of the body secreted by the kidneys by a process of filtration from blood called urination and excreted through the urethra. Cellular metabolism generates numerous waste compounds, many rich in nitrogen, that require elimination from the bloodstream...
, using the reaction of an
antibodyAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
or antibodies to its
antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
. The
assayAn assay is a procedure in molecular biology for testing and/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...
takes advantage of the specific binding of an
antibodyAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
to its
antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
.
Monoclonal antibodiesMonoclonal antibodies are monospecific antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent cell. Given almost any substance, it is possible to create monoclonal antibodies that specifically bind to that substance; they can then...
are often used as they only usually bind to one site of a particular molecule, and therefore provide a more specific and accurate test, which is less easily confused by the presence of other molecules. The antibodies picked must have a high affinity for the
antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
(if there is
antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
available, a very high proportion of it must bind to the
antibodyAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
).
Both the presence of
antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
or antibodies can be measured. For instance, when seeking to detect the presence of an infection the concentration of
antibodyAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
specific to that particular
pathogenA pathogen , infectious agent, or germ, is a biological agent that causes disease or illness to its host....
is measured. For measuring hormones such as
insulinInsulin is a hormone that has extensive effects on metabolism and other body functions, such as vascular compliance. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle, and stopping use of fat as an energy...
, the
insulinInsulin is a hormone that has extensive effects on metabolism and other body functions, such as vascular compliance. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle, and stopping use of fat as an energy...
acts as the
antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
.
For numerical results, the response of the fluid being measured must be compared to standards of a known concentration. This is usually done through the plotting of a standard curve on a graph, the position of the curve at response of the unknown is then examined, and so the quantity of the unknown found.
Detecting the quantity of
antibodyAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
or
antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
can be achieved by a variety of methods. One of the most common is to label either the
antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
or
antibodyAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
. The label may consist of an
enzymeEnzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, called the products. Almost all processes in a biological cell need enzymes to occur at...
(see enzyme immunoassay (EIA)),
colloidal goldColloidal gold, also known as "nanogold", is a suspension of sub-micrometre-sized particles of gold in a fluid — usually water. The liquid is usually either an intense red colour , or a dirty yellowish colour . The nanoparticles themselves can come in a variety of shapes...
(
lateral flow assaysLateral 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...
), radioisotopes such as I-125
RadioimmunoassayRadioimmunoassay is a very sensitive technique used to measure concentrations of antigens without the need to use a bioassay. It was developed by Rosalyn Yalow and Solomon Aaron Berson in the 1950s...
(RIA), magnetic labels (
magnetic 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...
- MIA) or
fluorescenceFluorescence is the emission of visible light by a substance that has absorbed light of a differing, usually invisible, wavelength. Absorption of a photon triggers the emission of a photon with a longer wavelength. A shorter wavelength emission is sometimes observed from multiple photon absorption...
. Other techniques include
agglutinationAgglutination is the clumping of particles. The word agglutination comes from the Latin agglutinare, meaning "to glue to."This occurs in biology in three main examples:...
,
nephelometryNephelometry is a technique used in immunology to determine levels of IgM, IgG, and IgA.It is performed by shining light on a sample, and measuring the amount of light scattered....
, turbidimetry and
Western BlotThe western blot is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide or by the 3-D structure of the protein...
.
Immunoassays have a particularly important role in the diagnosis of many infectious diseases, including
HIVHuman immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which the immune system begins to fail, leading to life-threatening opportunistic infections. Infection with HIV occurs by the transfer of blood, semen, vaginal fluid,...
. Immunoassays are just one type of diagnostic
HIV testHIV tests are used to detect the presence of the human immunodeficiency virus in serum, saliva, or urine. Such tests may detect HIV antibodies, antigens, or RNA.- Terminology :...
.
Types
Immunoassays can be divided into those that involve labelled reagents and those which involve non-labelled reagents. Those which involve labelled reagents are divided into homogenous and heterogeneous (which require an extra step to remove unbound
antibodyAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
or
antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
from the site, usually using a solid phase reagent) immunoassays.
Heterogeneous immunoassays can be competitive or non-competitive.
- In a competitive immunoassay, the antigen
An antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
in the unknown sample competes with labeled antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
to bind with antibodies. The amount of labeled antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
bound to the antibodyAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
site is then measured. In this method, the response will be inversely proportional to the concentration of antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
in the unknown. This is because the greater the response, the less antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
in the unknown was available to compete with the labeled antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
.
- In noncompetitive immunoassays, also referred to as the "sandwich assay," antigen
An antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
in the unknown is bound to the antibodyAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
site, then labeled antibodyAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
is bound to the antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
. The amount of labeled antibodyAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
on the site is then measured. Unlike the competitive method, the results of the noncompetitive method will be directly proportional to the concentration of the antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
. This is because labeled antibodyAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
will not bind if the antigenAn antigen is a substance that prompts the generation of antibodies and can cause an immune response. "Self" antigens are usually tolerated by the immune system; whereas "Non-self" antigens are identified as intruders and attacked by the immune system...
is not present in the unknown sample.
Because
homogeneous assays do not require this step, they are typically faster and easier to perform.
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