Immunoperoxidase
Encyclopedia
Immunoperoxidase is a type of immunostain
used in molecular biology
, medical research, and clinical diagnostics. In particular, immunoperoxidase reactions refer to a sub-class of immunohistochemical or immunocytochemical procedures in which the antibodies are visualized via a peroxidase-catalyzed reaction.
Immunohistochemistry
and immunocytochemistry
are methods used to determine in which cells or parts of cells a particular protein
or other macromolecule
are located. These stains use antibodies
to bind to specific antigen
s, usually of protein or glycoprotein
origin. Since antibodies are normally invisible, special strategies must be employed to detect these bound antibodies. In an immunoperoxidase procedure, an enzyme
known as a peroxidase
is used to catalyze
a chemical reaction
to produce a coloured product.
Simply, a very thin slice of tissue is fixed onto glass, incubated with antibody or a series of antibodies, the last of which is chemically linked to the peroxidase enzyme. After developing the stain by adding the chemical substrate
, the distribution of the stain can be examined by microscopy.
, i.e. produced in tissue culture. Monoclonal antibodies that consist of only one type of antibody tend to provide greater antigen specificity, and also tend to be more consistent between batches.
Optimal staining depends on a number of factors including the antibody dilution, the staining chemicals, the preparation and/or fixation of the cells/tissue, and length of incubation with antibody/staining reagents. These are often determined by trial and error
rather than any sort of systematic approach.
can be used instead of peroxidases for both direct and indirect staining methods. Alternatively, the primary antibody can be detected using fluorescent
label (immunofluorescence
), or be attached to colloidal gold particles for electron microscopy
.
In clinical diagnostics, immunostaining can be used on tissue biopsies for more detailed histopathological
study. In the case of cancer, it can aid in sub-classifying tumours. Immunostaining can also be used to help diagnose skin conditions, glomerulonephritis
and to sub classify amyloid
deposits. Related techniques are also useful in sub-typing lymphocyte
s which all look quite similar on light microscopy.
In laboratory research, antibodies against specific markers of cellular differentiation
can be used to label individual cell types. This can enable a better understanding of mechanistic changes to specific cell lineages resulting from a particular experimental intervention.
Immunostaining
Immunostaining is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample. The term immunostaining was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941...
used in molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
, medical research, and clinical diagnostics. In particular, immunoperoxidase reactions refer to a sub-class of immunohistochemical or immunocytochemical procedures in which the antibodies are visualized via a peroxidase-catalyzed reaction.
Immunohistochemistry
Immunohistochemistry
Immunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and...
and immunocytochemistry
Immunocytochemistry
Immunocytochemistry is a common laboratory technique that uses antibodies that target specific peptides or protein antigens in the cell via specific epitopes. These bound antibodies can then be detected using several different methods. ICC allows researchers to evaluate whether or not cells in a...
are methods used to determine in which cells or parts of cells a particular protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
or other macromolecule
Macromolecule
A macromolecule is a very large molecule commonly created by some form of polymerization. In biochemistry, the term is applied to the four conventional biopolymers , as well as non-polymeric molecules with large molecular mass such as macrocycles...
are located. These stains use antibodies
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 to specific 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, usually of protein or glycoprotein
Glycoprotein
Glycoproteins are proteins that contain oligosaccharide chains covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. In proteins that have segments extending...
origin. Since antibodies are normally invisible, special strategies must be employed to detect these bound antibodies. In an immunoperoxidase procedure, an enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
known as a peroxidase
Peroxidase
Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
is used to catalyze
Catalysis
Catalysis is the change in rate of a chemical reaction due to the participation of a substance called a catalyst. Unlike other reagents that participate in the chemical reaction, a catalyst is not consumed by the reaction itself. A catalyst may participate in multiple chemical transformations....
a chemical reaction
Chemical reaction
A chemical reaction is a process that leads to the transformation of one set of chemical substances to another. Chemical reactions can be either spontaneous, requiring no input of energy, or non-spontaneous, typically following the input of some type of energy, such as heat, light or electricity...
to produce a coloured product.
Simply, a very thin slice of tissue is fixed onto glass, incubated with antibody or a series of antibodies, the last of which is chemically linked to the peroxidase enzyme. After developing the stain by adding the chemical substrate
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
, the distribution of the stain can be examined by microscopy.
Types of antibodies
Originally all antibodies produced for immunostaining were polyclonal, i.e. raised by normal antibody reactions in animals such as horses or rabbits. Now, many are monoclonalMonoclonal antibodies
Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....
, i.e. produced in tissue culture. Monoclonal antibodies that consist of only one type of antibody tend to provide greater antigen specificity, and also tend to be more consistent between batches.
Methods for immunoperoxidase staining
The first step in immunoperoxidase staining is the binding of the specific (primary) antibody to the cell or tissue sample. The detection of the primary antibody can be then accomplished directly (example 1) or indirectly (examples 2 & 3).- Example 1. The primary antibody can be directly tagged with the peroxidasePeroxidasePeroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
enzyme which is then used to catalyse a chemical reaction to generate a coloured product.
- Example 2. The primary antibody can be tagged with a small molecule that can be recognised by a peroxidase-conjugated binding molecule with high affinity. The most common example of this is a biotinBiotinBiotin, also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin discovered by Bateman in 1916. It is composed of a ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...
linked primary antibody that binds to an enzyme-bound streptavidinStreptavidinStreptavidin is a 60000 dalton protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin . With a dissociation constant on the order of ≈10-14 mol/L, the binding of biotin to streptavidin is one of the strongest...
. This method can be used to amplifyAmplifierGenerally, an amplifier or simply amp, is a device for increasing the power of a signal.In popular use, the term usually describes an electronic amplifier, in which the input "signal" is usually a voltage or a current. In audio applications, amplifiers drive the loudspeakers used in PA systems to...
the signal.
- Example 3. An untagged primary antibody is detected using a general secondary antibody that recognises all antibodies originating from same animal species as the primary. The secondary antibody is tagged with the peroxidase enzyme.
Optimal staining depends on a number of factors including the antibody dilution, the staining chemicals, the preparation and/or fixation of the cells/tissue, and length of incubation with antibody/staining reagents. These are often determined by trial and error
Trial and error
Trial and error, or trial by error, is a general method of problem solving, fixing things, or for obtaining knowledge."Learning doesn't happen from failure itself but rather from analyzing the failure, making a change, and then trying again."...
rather than any sort of systematic approach.
Alternatives to peroxidase stains
Other catalytic enzymes such as alkaline phosphataseAlkaline phosphatase
Alkaline phosphatase is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation...
can be used instead of peroxidases for both direct and indirect staining methods. Alternatively, the primary antibody can be detected using fluorescent
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
label (immunofluorescence
Immunofluorescence
Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows...
), or be attached to colloidal gold particles for electron microscopy
Electron microscope
An electron microscope is a type of microscope that uses a beam of electrons to illuminate the specimen and produce a magnified image. Electron microscopes have a greater resolving power than a light-powered optical microscope, because electrons have wavelengths about 100,000 times shorter than...
.
Uses of immunoperoxidase staining
Immunoperoxidase staining is used in clinical diagnostics and in laboratory research.In clinical diagnostics, immunostaining can be used on tissue biopsies for more detailed histopathological
Histopathology
Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease...
study. In the case of cancer, it can aid in sub-classifying tumours. Immunostaining can also be used to help diagnose skin conditions, glomerulonephritis
Glomerulonephritis
Glomerulonephritis, also known as glomerular nephritis, abbreviated GN, is a renal disease characterized by inflammation of the glomeruli, or small blood vessels in the kidneys...
and to sub classify amyloid
Amyloid
Amyloids are insoluble fibrous protein aggregates sharing specific structural traits. Abnormal accumulation of amyloid in organs may lead to amyloidosis, and may play a role in various neurodegenerative diseases.-Definition:...
deposits. Related techniques are also useful in sub-typing lymphocyte
Lymphocyte
A lymphocyte is a type of white blood cell in the vertebrate immune system.Under the microscope, lymphocytes can be divided into large lymphocytes and small lymphocytes. Large granular lymphocytes include natural killer cells...
s which all look quite similar on light microscopy.
In laboratory research, antibodies against specific markers of cellular differentiation
Cellular differentiation
In developmental biology, cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type. Differentiation occurs numerous times during the development of a multicellular organism as the organism changes from a simple zygote to a complex system of...
can be used to label individual cell types. This can enable a better understanding of mechanistic changes to specific cell lineages resulting from a particular experimental intervention.