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Immunolabeling is a biochemical process that enables the detection and localization of an
antigen
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...
to a particular site within a cell, tissue, or organ. Antigens are organic molecules, usually
proteins
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
, capable of binding to an
antibody
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
. These antigens can be visualized using a combination of antigen-specific antibody as well as a means of detection, called a tag, that is covalently linked to the antibody. If the immunolabeling process is meant to reveal information about a cell or its substructures, the process is called
immunocytochemistry
Immunocytochemistry (ICC) is a common laboratory technique that is used to anatomically visualize the localization of a specific protein or antigen in cells by use of a specific primary antibody that binds to it. The primary antibody allows visual ...
.
Immunolabeling of larger structures is called
immunohistochemistry
Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to an ...
.
There are two complex steps in the manufacture of antibody for immunolabeling. The first is producing the antibody that binds specifically to the antigen of interest and the second is fusing the tag to the antibody. Since it is impractical to fuse a tag to every conceivable antigen-specific antibody, most immunolabeling processes use an indirect method of detection. This indirect method employs a
primary antibody
Primary and secondary antibodies are two groups of antibodies that are classified based on whether they bind to ''antigens or proteins'' directly or target another (primary) antibody that, in turn, is bound to an ''antigen or protein''.
Primary
...
that is antigen-specific and a
secondary antibody
Primary and secondary antibodies are two groups of antibodies that are classified based on whether they bind to ''antigens or proteins'' directly or target another (primary) antibody that, in turn, is bound to an ''antigen or protein''.
Primary
...
fused to a tag that specifically binds the primary antibody. This indirect approach permits mass production of secondary antibody that can be bought off the shelf.
Pursuant to this indirect method, the primary antibody is added to the test system. The primary antibody seeks out and binds to the target antigen. The tagged secondary antibody, designed to attach exclusively to the primary antibody, is subsequently added.
Typical tags include: a
fluorescent
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
compound, gold beads, a particular
epitope
An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The epitope is the specific piece of the antigen to which an antibody binds. The p ...
tag,
or an
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
that produces a colored compound. The association of the tags to the target via the antibodies provides for the identification and visualization of the antigen of interest in its native location in the tissue, such as the
cell membrane
The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment ( ...
,
cytoplasm
In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. The ...
, or
nuclear membrane
The nuclear envelope, also known as the nuclear membrane, is made up of two lipid bilayer membranes that in eukaryotic cells surround the nucleus, which encloses the genetic material.
The nuclear envelope consists of two lipid bilayer membrane ...
. Under certain conditions the method can be adapted to provide quantitative information.
Immunolabeling can be used in
pharmacology
Pharmacology is a branch of medicine, biology and pharmaceutical sciences concerned with drug or medication action, where a drug may be defined as any artificial, natural, or endogenous (from within the body) molecule which exerts a biochemica ...
,
molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
,
biochemistry
Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and ...
and any other field where it is important to know of the precise location of an antibody-bindable molecule.
Indirect vs. direct method
There are two methods involved in immunolabeling, the direct and the indirect methods. In the direct method of immunolabeling, the primary antibody is conjugated directly to the tag. The direct method is useful in minimizing
cross-reaction, a measure of nonspecificity that is inherent in all antibodies and that is multiplied with each additional antibody used to detect an antigen. However, the direct method is far less practical than the indirect method, and is not commonly used in laboratories, since the primary antibodies must be covalently labeled, which require an abundant supply of purified antibody. Also, the direct method is potentially far less sensitive than the indirect method.
Since several secondary antibodies are capable of binding to different parts, or domains, of a single primary antibody binding the target antigen, there is more tagged antibody associated with each antigen. More tag per antigen results in more signal per antigen.
Different indirect methods can be employed to achieve high degrees of specificity and sensitivity. First, two-step protocols are often used to avoid the cross-reaction between the immunolabeling of multiple
primary and secondary antibody mixtures, where secondary
fragment antigen-binding
The fragment antigen-binding region (Fab region) is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain. The variable domain contains the paratope (the ant ...
antibodies are frequently used. Secondly, haptenylated primary antibodies can be used, where the secondary antibody can recognize the associated
hapten
In immunology, 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 (in general, only large molecules, i ...
. The hapten is covalently linked to the primary antibody by
succinyl
Succinic acid () is a dicarboxylic acid with the chemical formula (CH2)2(CO2H)2. The name derives from Latin ''succinum'', meaning amber. In living organisms, succinic acid takes the form of an anion, succinate, which has multiple biological ro ...
imidesters or conjugated
IgG
Immunoglobulin G (Ig G) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each IgG ...
Fc-specific
Fab sections. Lastly, primary
monoclonal antibodies
A monoclonal antibody (mAb, more rarely called moAb) is an antibody produced from a cell Lineage made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell.
Monoclonal antibodies ca ...
that have different Ig isotypes can be detected by specific secondary antibodies that are against the
isotype of interest.
Antibody binding and specificity
Overall, antibodies must bind to the
antigens
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...
with a high specificity and affinity.
The specificity of the binding refers to an antibody's capacity to bind and only bind a single target antigen. Scientists commonly use
monoclonal antibodies
A monoclonal antibody (mAb, more rarely called moAb) is an antibody produced from a cell Lineage made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell.
Monoclonal antibodies ca ...
and
polyclonal antibodies
Polyclonal antibodies (pAbs) are antibodies that are secreted by different B cell lineages within the body (whereas monoclonal antibodies come from a single cell lineage). They are a collection of immunoglobulin molecules that react against a ...
, which are composed of synthetic peptides. During the manufacture of these antibodies, antigen specific antibodies are sequestered by attaching the antigenic peptide to an
affinity column and allowing nonspecific antibody to simply pass through the column. This decreases the likelihood that the antibodies will bind to an unwanted
epitope
An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The epitope is the specific piece of the antigen to which an antibody binds. The p ...
of the antigen not found on the initial peptide. Hence, the specificity of the antibody is established by the specific reaction with the protein or peptide that is used for immunization by specific methods, such as
immunoblotting
The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detect ...
or
immunoprecipitation
Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a samp ...
.
In establishing the specificity of antibodies, the key factor is the type of synthetic peptides or purified proteins being used. The lesser the specificity of the antibody, the greater the chance of visualizing something other than the target antigen. In the case of synthetic peptides, the advantage is the
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
sequence is easily accessible, but the peptides do not always resemble the 3-D structure or
post-translational modification
Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosome ...
found in the native form of the protein. Therefore, antibodies that are produced to work against a synthetic peptide may have problems with the native 3-D protein. These types of antibodies would lead to poor results in immunoprecipitation or
immunohistochemistry
Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to an ...
experiments, yet the antibodies may be capable of binding to the denatured form of the protein during an immunoblotting run. On the contrary, if the antibody works well for purified proteins in their native form and not
denatured, an immunoblot cannot be used as a standardized test to determine the specificity of the antibody binding, particularly in immunohistochemistry.
Specific immunolabeling techniques
Immunolabeling for light microscopy
Light
microscopy
Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of micr ...
is the use of a
light microscope
The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microsco ...
, which is an instrument that requires the usage of light to view the enlarged specimen. In general, a compound light microscope is frequently used, where two lenses, the eyepiece, and the
objective
Objective may refer to:
* Objective (optics), an element in a camera or microscope
* ''The Objective'', a 2008 science fiction horror film
* Objective pronoun, a personal pronoun that is used as a grammatical object
* Objective Productions, a Brit ...
work simultaneously to generate the magnification of the specimen.
Light microscopy frequently uses immunolabeling to observe targeted tissues or cells. For instance, a study was conducted to view the morphology and the production of hormones in pituitary adenoma cell cultures via light microscopy and other electron microscopic methods. This type of microscopy confirmed that the primary adenoma cell cultures keep their physiological characteristics ''in vitro'', which matched the histology inspection. Moreover, cell cultures of human pituitary adenomas were viewed by light microscopy and immunocytochemistry, where these cells were fixed and immunolabeled with a monoclonal mouse antibody against human GH and a polyclonal rabbit antibody against PRL. This is an example of how a immunolabeled cell culture of pituitary adenoma cells that were viewed via light microscopy and by other electron microscopy techniques can assist with the proper diagnosis of tumors.
Immunolabeling for electron microscopy
Electron microscopy
An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a hi ...
(EM) is a focused area of science that uses the
electron microscope
An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a hi ...
as a tool for viewing tissues.
Electron microscopy has a magnification level up to 2 million times, whereas light microscopy only has a magnification up to 1000-2000 times. There are two types of electron microscopes, the
transmission electron microscope
Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a gr ...
and the
scanning electron microscope.
Electron microscopy is a common method that uses the immunolabeling technique to view tagged tissues or cells. The electron microscope method follows many of the same concepts as immunolabeling for light microscopy, where the particular antibody is able to recognize the location of the antigen of interest and then be viewed by the electron microscope. The advantage of electron microscopy over light microscopy is the ability to view the targeted areas at their subcellular level. Generally, a heavy metal that is electron dense is used for EM, which can reflect the incident electrons. Immunolabeling is typically confirmed using the light microscope to assure the presence of the antigen and then followed up with the electron microscope.
Immunolabeling and electron microscopy are often used to view
chromosome
A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
s. A study was conducted to view possible improvements of immunolabeling chromosome structures, such as
topoisomerase IIα
DNA topoisomerase IIα is a human enzyme encoded by the ''TOP2A'' gene.
Topoisomerase IIα relives topological DNA stress during transcription, condenses chromosomes, and separates chromatids. It catalyzes the transient breaking and rejoining of ...
and
condensin
Condensins are large protein complexes that play a central role in chromosome assembly and segregation during mitosis and meiosis (Figure 1). Their subunits were originally identified as major components of mitotic chromosomes assembled in ''Xeno ...
in dissected
mitotic
In cell biology, mitosis () is a part of the cell cycle in which replicated chromosomes are separated into two new nuclei. Cell division by mitosis gives rise to genetically identical cells in which the total number of chromosomes is maintai ...
chromosomes. In particular, these investigators used UV irradiation of separated nuclei or showed how chromosomes assist by high levels of specific immunolabeling, which were viewed by electron microscopy.
Immunolabeling for transmission electron microscopy
Transmission electron microscopy
Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a g ...
(TEM) uses a transmission electron microscope to form a two-dimensional image by shooting electrons through a thin piece of tissue. The brighter certain areas are on the image, the more electrons that are able to move through the specimen.
Transmission Electron Microscopy has been used as a way to view immunolabeled tissues and cells. For instance, bacteria can be viewed by TEM when immunolabeling is applied. A study was conducted to examine the structures of CS3 and CS6
fimbriae in different ''Escherichia coli'' strains, which were detected by TEM followed by negative staining, and immunolabeling. More specifically, immunolabeling of the fimbriae confirmed the existence of different surface antigens.
Immunolabeling for scanning electron microscopy
Scanning electron microscopy (SEM) uses a scanning electron microscope, which produces large images that are perceived as three-dimensional when, in fact, they are not. This type of microscope concentrates a beam of electrons across a very small area (2-3 nm) of the specimen in order to produce electrons from said specimen. These secondary electrons are detected by a sensor, and the image of the specimen is generated over a certain time period.
Scanning electron microscopy is a frequently used immunolabeling technique. SEM is able to detect the surface of cellular components in high resolution. This immunolabeling technique is very similar to the immuno-fluorescence method, but a colloidal gold tag is used instead of a fluorophore. Overall, the concepts are very parallel in that an unconjugated primary antibody is used and sequentially followed by a tagged secondary antibody that works against the primary antibody.
Sometimes SEM in conjunction with gold particle immunolabeling is troublesome in regards to the particles and charges resolution under the electron beam; however, this resolution setback has been resolved by the improvement of the SEM instrumentation by backscattered electron imaging.
This is because electron backscattered diffraction patterns provide a clean surface of the sample to interact with the primary electron beam.
Immunolabeling with gold (Immunogold Labeling)
Immunolabeling with gold particles, also known as
immunogold staining, is used regularly with scanning electron microscopy and transmission electron microscopy to successfully identify the area within cells and tissues where antigens are located.
The gold particle labeling technique was first published by Faulk, W. and Taylor, G. when they were able to tag gold particles to anti-salmonella rabbit gamma globulins in one step in order to identify the location of the antigens of salmonella.
Studies have shown that the size of the gold particle must be enlarged (>40 nm) to view the cells in low magnification, but gold particles that are too large can decrease the efficiency of the binding of the gold tag. Scientists have concluded the usage of smaller gold particles (1-5 nm) should be enlarged and enhanced with silver. Although osmium tetroxide staining can scratch the silver, gold particle enhancement was found not to be susceptible to scratching by osmium tetroxide staining; therefore, many cell adhesion studies of different substrates can use the immunogold labeling mechanism via the enhancement of the gold particles.
Further Applications
Research has been conducted to test the compatibility of immunolabeling with fingerprints. Sometimes, fingerprints are not clear enough to recognize the ridge pattern. Immunolabeling may be a way for forensic personnel to narrow down who left the print. Researchers conducted a study which tested the compatibility of immunolabeling with many developmental techniques for fingerprints. They found that indanedione-zinc (IND-ZnCl), IND-ZnCl followed by
ninhydrin spraying (IND-NIN), physical developer (PD),
cyanoacrylate fuming (CA), cyanoacrylate followed by basic yellow staining (CA-BY), lumicyanoacrylate fuming (Lumi-CA) and polycyanoacrylate fuming (Poly-CA) all were compatible with immunolabeling.
Immunolabeling can not only extract donor profiling information from fingerprints, but can also enhance the quality of the fingerprints which both would be beneficial in a forensic case.
References
External links
{{Library resources box
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, by=no
, wikititle=Immunolabelling
, label=Immunolabelling
Labeling ProceduresMolecular cross-talk between the transcription, translation, and nonsense-mediated decay machineriesImmunolabeling as a tool for understanding the spatial distribution of fiber wall components and their biosynthetic enzymes
Medical diagnosis
Immunologic tests