Immunolabeling
![](http://upload.wikimedia.org/wikipedia/commons/thumb/6/67/Immunolabeling_process_image.png/220px-Immunolabeling_process_image.png)
Immunolabeling is a biochemical process that enables the detection and localization of 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 that is antigen-specific and a secondary antibody 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.[4] 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
Immunolabeling can be used in pharmacology, molecular biology, biochemistry and any other field where it is important to know of the precise location of an antibody-bindable molecule.[6][7][8]
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.[9] 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.[10] 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.[11]
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
Antibody binding and specificity
Overall, antibodies must bind to the
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
Specific immunolabeling techniques
Immunolabeling for light microscopy
Light
Immunolabeling for electron microscopy
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.[19]
Immunolabeling and electron microscopy are often used to view chromosomes. A study was conducted to view possible improvements of immunolabeling chromosome structures, such as topoisomerase IIα and condensin in dissected mitotic 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.[20]
Immunolabeling for transmission electron microscopy
Immunolabeling for scanning electron microscopy
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.[22] 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.[23] This is because electron backscattered diffraction patterns provide a clean surface of the sample to interact with the primary electron beam.[24]
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.[23] 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.[23][25]
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.[26]
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.[27] 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
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: CS1 maint: multiple names: authors list (link - ^ Kim, Oliver (2009-01-22). "Electron Microscopes vs. Optical (Light) Microscopes". Microbehunter Microscopy Magazine. Retrieved 2013-05-04.
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External links
- Labeling Procedures
- Molecular cross-talk between the transcription, translation, and nonsense-mediated decay machineries
- Nanoprobes Technical Help: Successful EM Immunolabeling
- Immunolabeling as a tool for understanding the spatial distribution of fiber wall components and their biosynthetic enzymes