|
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 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 fragment antigen-binding antibodies 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.〔 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Immunolabeling」の詳細全文を読む スポンサード リンク
|