Immunolabeling is a biochemical process that enables the detection and localization of an antigen to a particular site within a cell, tissue, or organ. Antigens are organic molecules, usually proteins, capable of binding to an antibody. 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. Immunolabeling of larger structures is called immunohistochemistry. 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. 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 compound, gold beads, a particular epitope tag, or an enzyme 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, cytoplasm, or nuclear of membrane. Under certain conditions the method can be adapted to provide quantitative information. 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.
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 antibodies Fab fragments are frequently used. Secondly, haptenylated primary antibodies can be used, where the secondary antibody can recognize the associated hapten. The hapten is covalently linked to the primary antibody by succinyl imidesters or conjugated IgG Fc-specific Fab sections. Lastly, primary monoclonal antibodies 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 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 and polyclonal antibodies, 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 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 or immunoprecipitation. 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 sequence is easily accessible, but the peptides do not always resemble the 3-D structure or post-translational modification 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 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
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